Penconazole and calcium improves drought stress tolerance and oil quality in canola

ABSTRACT

In this study, the effects of penconazole (PEN) and calcium (Ca) on growth amelioration and quality of seed oil in canola (Brassica napus L.) under drought stress were investigated. Drought stress reduced the growth parameters (fresh weight and dry weight) in canola; however the application of PEN and Ca improved these parameters under drought condition. Inducing effect of Ca on protein content was more prominent than PEN. Proline content increased under drought stress and PEN and Ca treatment caused more induction it under drought. PEN and Ca alleviated the negative effects of drought stress in canola by inducing antioxidant defense. The application of PEN and Ca caused a significant reduction in lipid peroxidation and hydrogen peroxide and mitigation of the drought induced oxidative stress. Drought stress induced protein content, total phenol, flavonol content, soluble sugar, palmitic acid and palmitoloic acid, and reduced flavonoid content, oleic acid, linolenic acid, and linolonoic acid in canola seed. PEN and Ca increased palmitic acid, linoleic acid (Omega-6), linolenic acid (Omega-3), oleic acid, protein, and soluble sugar. Our results indicated that application of PEN and Ca enables canola plants to withstand the deleterious impact of drought stress and caused improvement of antioxidant capacity, essential fatty acids (linolenic acid and linolonoic acid) and oil quality in canola seed.

KEYWORDS:

• Drought
• canola
• PEN
• Ca
• fatty acid

1. Introduction

Among the various abiotic stresses, drought stress is a more severe constraint that reduces the yield and quality of crop plants (Yamaguchi-Shinozaki et al. 2002; Hongbo et al. 2005). Reduce the product by drought has been reported in many crop species, which depends on the severity and duration of the stress period. There is an urgent need to improve the compatibility of crop species to drought stress to maintain high yield and quality (Kaczmarek et al. 2017).

Among the major food crops, Brassica crops are more susceptible to drought and salinity because they grow mainly in arid and semiarid areas. Canola (Brassica napus L.) is the third source of plant oils after Palm and soybean in the world that its seed contains about 40–44% oil (Carmody 2001). Remaining of canola after extraction of oil contains 38–44% high-quality protein that is used for animal nutrition (Walker and Booth 2007). Canola oil has potential for developing biodiesel market. Canola is relatively poorly adapted to drought stress and its yield is reduced (Wright et al. 1997). Seed yield reduced 19–39% as compared to control, when drought stress was imposed at reproductive stage (Gunasekara et al. 2006).

Penconazole (PEN) [1-(2,4-dichloro-β-propylphenethyl)-1 H-1,2,4-triazole] is a triazole compound. Triazoles are fungicides that have the characteristics of growth regulating and stress protecting (Jaleel et al. 2007a). Triazole compounds cause various morphological and biochemical responses in plants including stimulation of root growth, reduction in free-radical damage, and increase in antioxidant capacity (Jaleel et al. 2007a; Manivannan et al. 2007).

Calcium (Ca²⁺) is an essential element for plants and causes initiation of bud formation, polar growth, gas-exchange regulation, secretion, movements and light- and hormone-regulated growth and development (Hepler and Wayne 1985). Calcium mitigated the adverse effects of drought stress in plants (Jaleel et al. 2007b), and is involved in signaling responses against drought (Shao et al. 2008). Calcium has a central role in many defense mechanisms under drought, and Ca²⁺ signaling is required for drought tolerance or resistance (Cousson 2009).

There are many reports that show that application of PEN and Ca can play a major role in increasing plant tolerance against abiotic stresses (Merati et al. 2014; Upadhyaya et al. 2011). There are no reports regarding the effect of PEN and Ca on growth, physiological and biochemical parameters and oil fatty acid composition in canola. Thus, this work targets to investigate improvement effects of PEN and calcium on canola under drought stress. Moreover, deciphering the physiological biochemical mechanisms responsible for growth promotion and too, the effect of PEN and Ca on fatty acid composition, oil quality and different compounds in canola seed are other aims of the present study.

2. Materials and methods

2.1. Plant materials and growth parameters

Seeds of canola (B. napus L.) were obtained from the Seed and Plant Improvement Research Institute, Karaj, Iran. Seeds of canola were sown in each plastic pot containing an equal mixture of peat and perlite. After germination, the seedlings were thinned to five plants per pot. Plants were grown at average day/night temperatures of 25/18°C. The pots were irrigated with equal amount of half strength Hoagland solution for four week. Drought stress was imposed by PEG (5% (W/V) of PEG 6000) for three week. In the preliminary experiments, 5, 10, 15, and 20 mg l⁻¹ PEN and 5, 10, 15, and 20 mM CaCl₂ were used to determine the optimum concentrations. According to growth parameters and based on the studied PEN and CaCl₂ concentrations, 15 mg l⁻¹ of PEN and 15 mM of CaCl₂ were selected as the optimum concentrations for further studies. To determine the effect of PEN and Ca, PEG-treated plants were supplemented with 15 mg l⁻¹ of PEN or 15 mM of CaCl₂. The treatments were as follows: (1) Control: without PEG, CaCl₂ and PEN, (2) PEG, (3) PEG and PEN, (4) PEG and CaCl₂. Aqueous calcium chloride solution and PEN was applied uniformly to the plants once a week for 3 weeks. Each treatment was carried out in triplicate. Three weeks after treatment, plants were collected for analyses in all the experiments. The plants were evaluated 3 weeks after drought stress in terms of fresh weight (FW) and dry weight (DW).

2.2. Protein and proline content

Protein content was measured according to Bradford (1976), using bovine serum albumin (BSA) as standard. Proline content was determined following Bates et al. (1973). Plant material (0.05 g) was homogenized with 5 ml of 3% sulfosalicylic acid and the homogenate was centrifuged at 13,249 × g for 20 min. The resulting solution (2 ml) was treated with 2 ml acid ninhydrin and 2 ml of concentrated glacial acetic acid, shaken vigorously, and boiled for 1 h at 100°C. The reaction mixture was extracted with 4 ml toluene, and the absorbance was measured at 520 nm by UV – visible spectrophotometer (Shimadzu UV-160, Tokyo, Japan). The content of proline was determined using L-proline as standard.

2.3. Hydrogen peroxide and malondialdehyde content

The content of hydrogen peroxide (H₂O₂) was estimated according to method of Velikova et al. (2000). The plant material (0.5 g) was homogenized in an ice bath with 5 ml of 0.1%(w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 11,289 × g for 15 min and 0.5 ml of the supernatant was added to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1M of potassium iodide (KI). The absorbance of the supernatant was measured at 390 nm. The content of H₂O₂ was calculated by comparison with a standard curve made by using different concentrations of H₂O₂. The lipid peroxidation was determined based on malondialdehyde (MDA) content by Heath and Packer method (1968). Plant material (0.5 g) was homogenized in 5 ml of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 7840 × g for 20 min. To 1 ml aliquot of the supernatant, 4 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA was added. The mixture was heated at 95ºC for 30 min and quickly cooled in an ice bath. After centrifugation at 7840 × g for 15 min, the absorbance of the supernatant was recorded at 532 and 600 nm. The content of MDA was calculated using extinction coefficient of 155 mM⁻¹cm⁻¹.

2.4. Peroxidase, superoxide dismutase, and DPPH-radical scavenging activity

Peroxidase (POX) activity was measured according to the method of Abeles and Biles (1991). The reaction mixture contained 2 ml of 0.2 M acetate buffer (pH 4.8), 0.2 ml H₂O₂ (3%), 0.1 ml 40 mM benzidine, and 0.1 ml enzyme extract. The increase in absorbance was recorded at 530 nm. Superoxide dismutase (SOD) activity was estimated by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) as described by Giannopolitis and Ries (1977) in a reaction mixture containing 50 mM sodium phosphate buffer (pH 7.5), 13 mM methionine, 75 μM NBT, 75 μM riboflavin, 0.1 mM EDTA, and 100 μl of enzyme extract. The reaction mixture was irradiated for 16 min and absorbance was read at 560 nm against the nonirradiated blank. For estimation of DPPH (1, 1-diphenyl-2- picrylhydrazyl) radical scavenging activity used of Abe et al. (1998) method. Leaf material (100 mg) was homogenized in 1 ml of 96% ethanol and then insoluble materials removed by centrifuge at 3500 × g for 5 min. 20 μl of extracting solution was mixed with 800 μl of DPPH (0.5 mM in ethanol). The absorbance of the resulting solution was measured at 517 nm after 30 min in darkness. Using the following equation the free radical scavenging activity was calculated:

2.5. Total phenol and flavonol content

For determination of total phenol content 0.1 g of seed was extracted with boiling 80% methanol for 3 h according to Conde et al. (1995). Total phenol content was determined by using Folin-Ciocalteu reagent according to Akkol et al. (2008). One milliliter of methanolic extract was mixed with 5 ml Folin-Ciocalteu reagent and 4 ml sodium carbonate solution 7.0%. The mixtures were allowed to stand for 2 h before its absorbance was measured at 765 nm. Gallic acid was used as a standard for the calibration curve. Flavonol content was determined according to Akkol et al. (2008). One ml of methanolic extract, 3 ml of sodium acetate, and 1 ml of aluminum chloride solution were mixed and absorbance was measured at 445 nm after 2.5 h and rutin was used as a standard.

2.6. Flavonoid and anthocyanin content

Approximately 0.1 g of seed was homogenized in 3 ml of methanol. Flavonoid content was determined using aluminum chloride colorimetric method. Methanolic extract (0.5 ml) was mixed with 1.5 ml of pure methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water and kept at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm and expressed in µg g⁻¹ fresh weight (Chang et al. 2002). Anthocyanin content was determined in 0.3% HCl in methanol at 25°C using the extinction coefficient (33,000 cm² mol⁻¹) at 550 nm (Wagner 1979).

2.7. Tocopherol and soluble sugar content

Tocopherol content was estimated according to the method of Kayden et al. (1973). Approximately 0.1 g of seed was homogenized in 3 ml of ethanol. One ml of the ethanol extract was pipetted in to a 4 ml glass test tube. The extract was mixed with 0.2 ml of 0.2% bathophenanthroline in ethanol and the content of each tube were homogenized. The assay proceeded rapidly from this point and care was taken not to expose the solutions to direct light. 0.2 ml of a 1 mM FeC1₃ solution in ethanol was added, followed by mixing with a vortex mixer. After 1 min, 0.2 ml of 1 mM H₃PO₄ solution in ethanol was added and the contents of the tubes were again mixed. The absorbance of the solutions was determined at 534 nm using α-tocopherol as standard. Soluble sugar content was determined based on the method of phenol sulfuric acid using glucose as a standard. The seed (0.1 g) was extracted using 3 ml deionize water by mortar and pestle. To determine soluble sugar content, 50 µl of extract was mixed with 450 µl of water and 500 µl of 5% phenol solution and 2.5 ml of concentrated sulfuric acid were added rapidly so that the solutions were well mixed and allowed to stand at room temperature for 30 min. The absorbance of the samples was measured at 485 nm (Dubois et al. 1956).

2.8. Fatty acid analyses

Approximately 0.1 g of seed was placed in test tubes. One milliliter of transesterification reagent (methanol/acetyl chloride, 20:1 v/v) was added to each tube. The tubes were heated at 100°C for 1 h for the transmethylation, being shaken every 10–15 min. The mixture was cooled to room temperature, and 1 ml each of water and hexane were added. The tubes were shaken and centrifuged. The upper one (hexane) was transferred to another tube (Rodriguez-Ruiz et al. 1998) and fatty acid analyzed by gas liquid chromatography. Calculations were also made for oleic desaturation ratio (ODR), linoleic desaturation ratio (LDR), and oleic/linoleic acid ratio (OLR) (Pleines and Friedt 1988).

2.9. Statistical analysis

Each experiment was repeated three times and the data were analyzed by using one -way analysis of variance (ANOVA) using SPSS (version 21) and means were compared by Duncan’s test at the 0.05 level of confidence. PCA (principal component analysis) and HCA (Hierarchical cluster analysis) were used for evaluating correlation between each pair of variables and performed using XLSTAT (2016) and online CIMminner software, respectively.

3. Results

Growth parameters (FW and DW) significantly decreased under drought (Fig. 1). FW and DW decreased 36.44% and 36.59% as compared to control, respectively. PEN and Ca treatment increased FW and DW in canola under drought stress. PEN and Ca caused an increase of 39.67% and 39.43% in DW, respectively.

Figure 1. Fresh weight (A) and Dry weight (B) in canola plant and protein content (C) and soluble sugar content (D) in canola seed under drought, PEN, and Ca treatment. Vertical bars indicate Means ± SE based on three replicates. Different letters above columns indicated significant (P < 0.05) differences.

Drought caused no significant change in protein content, but PEN and Ca treatment increased it. Inducing effect of Ca on protein content was more prominent than PEN. Drought stress increased (49.25%) in proline content as compared to control. PEN and Ca treatment caused more induction proline content under drought. Drought significantly increased H₂O₂ content. By contrast, the application of PEN and Ca decreased this parameter by 29.9% and 60.69%, respectively, compared to drought-stressed plants. MDA content was enhanced by drought stress, but PEN treatment decreased it, compared to drought-stressed plants. Drought and Ca did not induce any changes in POX activity as compared to control. PEN treatment increased POX activity in canola under drought. Drought increased SOD activity, but PEN and Ca treatment decreased it. DPPH radical scavenging activity decreased under drought stress. PEN and Ca treatment induced DPPH radical scavenging activity, but this induction was higher by Ca (Table 1).

Table 1. Effects of PEN and Ca on different parameters in canola leaf under drought.

Treatment	Protein (mg g^−1FW)	Proline (µg g^−1FW)	H2O2 (µmol g^−1FW)	MDA (ng g^−1FW)	POX (U mg^−1 protein)	SOD (U mg^−1 protein)	DPPH (%)
Control	251.2 ± 10.2 c	655.16 ± 14 d	5.417 ± 0.8 c	1.17 ± 0.09 b	0.0210 ± 0.001 b	0.176 ± 0.009 b	51.45 ± 2.56 b
Drought	239.346 ± 12 c	1291.01 ± 12 c	12.094 ± 1.2 a	1.53 ± 0.13 a	0.0170 ± 0.003 b	0.190 ± 0.01 a	24.51 ± 6.12 c
Drought+ PEN	319.786 ± 8.9 b	1689.16 ± b	8.477 ± 1.56 b	1.29 ± 0.21 b	0.0487 ± 0.001 a	0.178 ± 0.005 b	64.80 ± 5.89 b
Drought+ Ca	448.84 ± 11.5 a	2023.12 ± a	4.754 ± 0.56 d	1.57 ± 0.14 a	0.0175 ± 0.006 b	0.109 ± 0.02 c	95.06 ± 7.98 a

Values are means ± SE of three replicates. Different letters indicated significant (P < 0.05) differences.

Drought, PEN and Ca treatment increased protein content in canola seed. In contrast to drought, PEN and Ca treatment increased soluble sugars content in canola seed and too, increasing effect of Ca was higher (Fig. 1C,D).

Total phenol content was enhanced in seed by drought. Ca treatment increased total phenol content in seed but PEN treatment decreased it under drought stress. Drought stress significantly increased (44.40%) favonol content in canola seed, but application of PEN and Ca reduced this parameter. Drought and Drought in combination to PEN caused a significant reduce in flavonoid content in canola seed, but Ca treatment induced it. Anthocyanin content in canola seed was showed any significant change by drought and PEN. Application of Ca decreased anthocyanin content in canola seed (Fig. 2).

Figure 2. The effect of drought stress, PEN, and Ca on total phenol (A), flavonol (B), flavonoid (C), and anthocyanin (D) in canola seed. Vertical bars indicate mean ± SE of three replicates. Different letters indicate significant differences at P < 0.05.

The application of Ca increased tocopherol content in canola seed by 68.47% compared to control. All treatment (drought, PEN, and Ca) caused non-significant change in DPPH radical scavenging activity in canola seed (Fig. 3A,B).

Figure 3. The effect of PEN, Ca, and PEG on tocopherol content (A), inhibition of DPPH radical (B), palmitic acid (C), and palmitoloic acid (D) in canola seed. Vertical bars indicate mean ± SE of three replicates. Different letters indicate significant differences at P < 0.05.

Drought, PEN, and Ca treatment enhanced palmitic acid (C₁₆) and palmitoloic acid (C_16:1) in canola seed as compared to control. Stearic acid (C₁₈) in seed was not showed a significant change under all treatment. In contrast to drought, PEN and Ca treatment increased oleic acid (C_18:1). Drought stress reduced linoleic acid (C_18:2) in canola seed, but exogenous application of PEN and Ca increased it as compared to control. PEN and Ca caused a significant induce linolenic acid (C_18:3) in canola seed as compared to stressed- plants. PEN and Ca treatment increased ODR and LDR in canola seed. Drought in combination to Ca decreased OLR (Figs. 4,5).

Figure 4. Effect of PEN and Ca on stearic acid (A), oleic acid (B), linoleic acid (C), and linolenic acid (D) in canola seed under drought. Vertical bars indicate mean ± SE of three replicates. Different letters above columns indicated significant (P < 0.05) differences.

Figure 5. Impact of PEG, PEN, and Ca on ODR (A), LDR (B), and OLR (C) in canola seed. Columns indicate mean ± SE based on three replicates. Means with different letters for each cultivar indicate a significant difference at P < 0.05 using Duncan multiple range test.

3.1. PCA analyses

According to correlation analyses based on Pearson’s coefficient in canola, DW exhibited positive correlations with POX, DPPH (Leaf), protein, and proline, but it showed a negative correlation with H₂O₂ and MDA. H₂O₂ and MDA displayed positive correlations to each other. H₂O₂ exhibited negative correlation with protein and DPPH (Leaf). MDA showed a negative correlation with POX and SOD. DW and FW showed a positive correlation with linoleic acid, linolenic acid, ODR, LDR, DPPH (Seed), and soluble sugar, but they showed a negative correlation with palmitic acid, palmitoloic acid, OLR, total phenol, and flavonol. Palmitic acid and palmitoloic acid displayed positive correlations to each other, but they showed a negative correlation with linolenic acid, ODR and LDR. Positive correlations were obtained between protein, soluble sugar, total phenol, tocopherol, and oleic acid in canola seed (Fig. 6).

Figure 6. Loading plots of principle components 1 and 2 of the PCA (A) and Heatmap (B) of physiological and biochemical changes in canola.

4. Discussion

Water shortage is an environmental limitation for the production of plant. Reduction of yield by drought is more than reducing other factors. Growth occurs through cell division, cell enlargement and differentiation, and includes genetic, physiological, ecological and morphological events and their complex interactions. The quality and quantity of plant growth depend on these events, which are affected by water shortage. Cell growth is one of the most drought-sensitive physiological processes due to the reduction in turgor pressure (Taiz and Zeiger 2006). Three main mechanisms decline yield by drought stress: (i) reduced canopy absorption of photosynthetic active radiation, (ii) reduce the efficiency of radiation use, and (iii) decreased harvest index (Earl and Davis 2003).

The present investigation effectively confirmed that PEN and Ca could induce the drought tolerance in canola. Triazoles by change in hormonal statue cause increase dry weight under drought stress (Grossman 1990). Triazole enhanced the whole plant dry weight in rose (Jenks et al. 2001). Some studies have shown that CaCl₂ can play an important role to ameliorate the negative effects of drought stress on several species. These responses could be attributed to that Ca is a crucial secondary messenger in the signaling processes to many defense mechanisms that are induced by drought stress (Xiang et al. 2008; Shao et al. 2008). Increasing plant growth as a response to CaCl₂ occurs as a result of cell elongation and cell division (Tanimoto 1990; Hirschi 2004).

Plants accumulate different types of organic and inorganic solutes in the cytosol to lower osmotic potential and maintain cell turgor (Rhodes and Samaras 1994). Protein accumulation is used as osmotic regulation which increases plant’s tolerance to adverse effects of abiotic stresses (Ma and Turner 2006). The accumulation of proline regulates protein turnover and stress protective proteins and too, is involved in ROS scavenging (Ahmad et al. 2015). Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell proliferation or cell death, and trigger specific gene expression (Szabados and Savoure 2009). Accumulation of protein and proline by PEN and Ca indicates the positive role of PEN and Ca in mitigating the negative impact of drought stress in canola. Triazole induces a temporary increase in ABA content. Increase of ABA content can be due to increased proline and amino acid content in triazole-treated carrot plants (Gopi et al. 2007). Paclobutrazol increased the proline content in Eruca sativa seedlings (Mathur and Bohra 1992). Calcium increased proline by breakdown of proline-rich protein and synthesis of proline and amino acids (Jun et al. 2000).

The various types of stresses (salt stress, drought, temperature, heavy metals, pollution, high irradiance, pathogen in fection, etc.) disturbed in balance between ROS generation and ROS scavenging. ROS react with unsaturated fatty acids and cause peroxidation of membrane lipids, leakage of cellular contents, rapid desiccation, and cell death. The ability of plants to induce enzymatic defense against uncontrolled lipid peroxidation is an important trait of the tolerance mechanisms (Jaleel et al. 2007a; Velikova et al. 2000). The scavenging of ROS is required to maintain metabolic functions under stress. ROS scavenging depends on detoxification mechanism provided by enzymatic and non-enzymatic antioxidants. SOD belongs to the family of metalloenzymes in all aerobic organisms and is the first line of defense against ROS-induced damages under abiotic stresses. The SOD catalyzes the removal of O₂^•− by dismutating it in to O₂ and H₂O₂ and POX scavenge H₂O₂ in to H₂O (Mittler 2002). PEN and Ca application caused an increase in DPPH-radical scavenging activity and decreased MDA and H₂O₂ content which suggested a protective role of PEN and Ca in mitigating oxidative stress in canola. Triazoles induced the antioxidant capacity in oxidative stressed plants as compared to control (Sankar et al. 2007). Increase in antioxidants cause scavenging of harmful free radicals, inhibition of electrolyte leakage and lipid peroxidation that correlated with ability of triazoles in maintaining the membrane integrity (Fletcher et al. 2000). This stress-ameliorating role of calcium can be attributed to the enhancement of antioxidant activity, which was shown to decrease lipid peroxidation in calcium-treated plants (Asgher et al. 2014).

Canola has the most phenolic compounds (about tenfold) in comparison to the other oilseeds (soybean, sunflower) (Naczk et al. 1998). Canola phenolic compounds are including sinapinic acid and other derivatives which have high antioxidant activity (Szydłowska-Czerniak et al. 2011). The main unsaponifiable components in canola oil are tocopherols and phytosterols. Tocopherols (vitamin E) are known to be very efficient natural antioxidants (Yanishlieva-Maslarova et al. 2001). Moreover, antioxidant compounds in canola including polyphenols, sterols, flavonoids, tocopherols etc. have an important role in prevent of some chronic diseases, such as heart, neurodegenerative, aging, cancer, and rheumatoid arthritis (Amarowicz et al. 2003; Kozlowska et al. 1983; Siger et al. 2004). Canola oil consists of four important fatty acids: palmitic acid, linoleic acid, linolenic acid, and oleic acid. Linolenic acid in canola oil is more than the other plant oils. Canola oil is high in oleic acid which is commonly used for food and industrial purposes and this edible oil is very healthy (Zhang et al. 2004). Canola is a major oil crop and also supplies proteins for the feed. Proteins showed the major category of storage compounds and their amino acid composition was found to be very close to that of canola in different conditions. In fact, oil-free canola meal contains 38–40% of crude proteins that exhibit an amino acid composition with high levels of essential sulfur containing amino acids (Nesi et al. 2008). Mingeau (1974) showed that adverse effects of drought stress on oil biosynthesis were more than on protein biosynthesis. Jensen et al. (1996) observed that, drought stress had opposing effects on oil and protein content of B. napus seeds at vegetative stage of growth. Bouchereau et al. (1996) showed that drought stress, during the flowering stage, affected the oil content and fatty acid composition of canola seeds. Mekki et al. (1999) also showed that drought stress increased the percentage of palmitic acid and reduced unsaturated fatty acids. Pham-Thi et al. (1986) reported that water stress decreased the degree of fatty acids unsaturation which was attributed to the inhibition in the biosynthesis of polyunsaturated fatty acids and suppression in the activities of desaturases. Triazole compounds caused an increase in oil content in Helianthus annuus L. (Sunflower) under drought stress (Rabert et al. 2015). Exogenous of calcium mitigated the adverse effects of NaCl stress in pea plants and increased the yield components (Howladar and Rady 2012). Ca treatment increased total phenol, tocopherol and flavonoid in canola seed. PEN and Ca enhanced palmitic acid, linoleic acid (Omega-6), linolenic acid (Omega-3), oleic acid, protein, and soluble sugar. Overall, PEN and Ca caused an increase in antioxidant capacity, nutritional value, and improvement of quality in canola seed.

In conclusion, PEN and Ca helped to resistance of canola to drought stress and increased plant growth. Such positive effects of PEN and Ca can be due to their impact on improvement of antioxidant defense systems, accumulation of protein and proline and declined in H₂O₂ and MDA content. Thus, PEN and Ca caused improvement of growth and increase antioxidant capacity, nutritional value, and quality in canola seed.

Author contribution

Maryam Rezayian has contributed in the major bench experiments. Dr. Vahid Niknam and Dr. Hassan Ebrahimzadeh equally designed the experiments. All authors read and approved the manuscript.

Additional information

Funding

The financial support of this research was provided by College of Science, University of Tehran.