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Nitric oxide regulates oxidative defense system, key metabolites and growth of broccoli (Brassica oleracea L.) plants under water limited conditions
Scientia Horticulturae 254 (2019) 7–13
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Nitric oxide regulates oxidative defense system, key metabolites and growth of broccoli (Brassica oleracea L.) plants under water limited conditions
T
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Aneeqa Munawara, Nudrat Aisha Akrama, , Abrar Ahmada, Muhammad Ashrafb a b
Department of Botany, Government College University, Faisalabad, Pakistan University of Agriculture Faisalabad, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: Broccoli (Brassica oleracea) Drought stress Nitric oxide Antioxidants
Nitric oxide (NO) is a diffusible gaseous molecule and has been under wide consideration because of its ability to mitigate adverse effects of several abiotic stresses on plants. In the current study, it was determined whether or not exogenous application (presowing seed treatment and foliar application) of sodium nitroprusside (SNP), donor of nitric oxide (NO), could alleviate the drastic effects of drought stress on broccoli plants. The broccoli seeds were soaked in 0.02 mM NO solution or distilled water for pre-sowing and control treatments, respectively. Two levels of water stress (control, 100% field capacity (FC) and 60% FC) were applied to 4 week-old broccoli (Brassica oleracea L.) plants. Foliar treatment of NO (0.02 mM) was applied to broccoli plants after 3 weeks of initiation of drought stress. After 12 days of foliar application, leaf samples were collected to determine photosynthetic and antioxidant activities as well as other biochemical parameters. The results showed that water deficit conditions decreased the shoot fresh and dry weights and shoot length, glycine betaine, and chlorophyll contents, while it enhanced ascorbic acid (AsA), hydrogen peroxide and activities of CAT and SOD enzymes. However, exogenously applied NO as a presowing seed treatment or foliar spray enhanced the fresh and dry biomass of shoot, shoot length, chlorophyll contents, GB, total phenolics, total soluble proteins and activities of SOD and POD enzymes in broccoli plants under water deficiency. It was also observed that foliar application of NO was more effective in enhancing the drought tolerance in broccoli plants as compared to pre-sowing application of NO. Therefore, foliar as well as pre-sowing application of NO could be helpful in up-regulating the oxidative defense system of broccoli plants under water deficit conditions.
1. Introduction Plant growth restriction is a ubiquitous phenomenon in agricultural food production in many parts of the world, mainly due to various environmental stresses (Hatamian et al., 2018; Souri and Hatamian, 2019). Abiotic stress such as drought stress has drastic effects on the development, growth and yield of plants (Wani et al., 2016). Khan et al. (2010) reported that water deficit stress reduces the mean productivity of grain crops from 17 to 70%. Drought stress reduces plant water content, nutrient uptake, leaf size, stem elongation, root proliferation, gas exchange, water use efficiency, transpiration rate, germination rate, cell division and expansion (Farooq et al., 2009; Galahitigama and Wathugala, 2016). Drought stress impairs the enzyme activity and decreases the energy supply (Farooq et al., 2009). Drought stress results into impaired stomatal conductance, turgor loss and nutritional imbalance (Galahitigama and Wathugala, 2016). Drought stress decreases the seed production by plants as it severely affects pollination (Asrar
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and Elhindi, 2011). Water deficit also causes oxidative stress which damages the cellular membranes and impairs photosynthesis (Aziz et al., 2018). Many studies have used various organic or mineral compounds to alleviate adverse effects of abiotic stresses on plant production (Souri and Hatamian, 2019). To alleviate the adverse effects of drought stress on plants, a number of means are in practice these days. Of these, exogenous application of a variety of plant growth regulators (PGRs) are being used to improve stress tolerance in plants (Akram et al., 2018). Different nitrogen compounds including various amino acids, peptides and nitric oxide have been shown to reduce the adverse effects of abiotic stresses on plants (Souri and Hatamian, 2019). Recently, it has been shown that ornamental judas tree (Cercis siliquastrum) have better tolerance to lead and cadmium toxicity under higher nitrate application in irrigation water (Hatamian et al., 2018). Nitric oxide (NO) is contemplated as one of the potential PGRs which can effectively offset the adverse effects of different abiotic stresses including drought
Corresponding author. E-mail address: [email protected] (N.A. Akram).
https://doi.org/10.1016/j.scienta.2019.04.072 Received 3 January 2019; Received in revised form 25 April 2019; Accepted 26 April 2019 Available online 03 May 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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2.2. Chlorophyll contents
stress. Nitric oxide is a gas which acts as a signaling molecule in plants under abiotic stress conditions (Boogar et al., 2014). Nitric oxide is known to break seed dormancy and promote germination (Krasuska et al., 2015) as well as it stimulates growth of root hairs (Lombardo et al., 2006), lateral and adventitious roots (Fernández-Marcos et al., 2011). Several reports have shown that NO is involved in the maintenance of chlorophyll contents (Liu and Guo, 2013), stomatal movements (Chen et al., 2013), vegetative growth (Lozano-Juste and León, 2011), homeostasis of iron (Buet and Simontacchi, 2015), production of nodules by symbiosis (Hichri et al., 2015), and hormonal metabolism (Sanz et al., 2015). Nitric oxide can also scavenge ROS and calcium signaling thereby improving the immunity of plants (Simontacchi et al., 2015). It has been reported that like other chemicals, sodium nitroprusside as a donor of NO is being used exogenously to mitigate the adverse effects of different abiotic stresses including drought stress (Boogar et al., 2014). Broccoli (Brassica oleracea L.) is considered as one of the major vegetables (López-Berenguer et al., 2006) and is cultivated throughout the world (Sotelo et al., 2014). It has high contents of minerals and vitamins and has beneficial effects on the health of human beings (Jeffery and Araya, 2009). Broccoli has anti-cancerous properties (De Figueiredo et al., 2015) as it is a good source of glycosinolates, fibers and minerals such as Fe, K, etc, vitamin C and K (Yuan et al., 2009; Ravikumar, 2015). The main objective of this study was to determine whether or not exogenously applied sodium nitroprusside, donor of NO, as a presowing seed treatment or foliar application could mitigate the adverse effects of drought stress on broccoli plants particularly with relation to the oxidative defense system.
Chlorophyll contents were measured using the method of Arnon (1949). Fresh leaf sample (0.5 g) was dipped into 10 ml acetone (80%) and allowed it to stand for one night in a cool place. Chlorophyll a and b contents were determined using a spectrophotometer. 2.3. Leaf free proline It was determined according to the method of Bates et al. (1973). Leaf samples (0.5 g) were ground in 10 ml of sulfosalicylic acid (3%) solution and filtered. The filtrate (2 ml) of each sample was taken into a separate labeled test tubes, and added acidic ninhydrin (1.26 g ninhydrin + 30 ml glacial acetic acid + 20 ml 6 M ortho-phosphoric acid) solution (2 ml) and glacial acetic acid (2 ml). Later on, the mixture incubated at 100 °C for 60 min. After that, toluene (4 ml) was added to each test tube and all tubes were shaken well. The absorbance of the treated samples was read at 520 nm using a spectrophotometer. 2.4. Glycine betaine (GB) GB in leaf tissue was determined following the Grieve and Grattan (1983) method. Leaf sample (0.5 g) was extracted in 10 ml distilled water and allowed it to stay at 4 °C for one night. After that, these samples were centrifuged at 10,000 rpm for 10 min. The supernatant (1 ml) from each sample was taken in a separate labeled test tube and added 1 ml of H2SO4 (2N) followed by 0.2 ml KI3 (1 M). All these test tubes were placed in an ice bath and allowed them to cool for one and half hour. Later on, 2.8 ml distilled water followed by 1–2 dichloroethane (6 ml) were added to them. Two layers were formed, discarded the upper layer and used the lower layer for taking readings at 365 nm using a spectrophotometer.
2. Materials and methods This study was conducted to evaluate the role of different modes (pre-sowing and foliar) of exogenously applied nitric oxide (NO) in improving drought tolerance of broccoli (Brassica oleracea L.). An experiment was conducted in the research area of GC University Faisalabad, Pakistan during November-January. During this period, average climate conditions were recorded as: temperature, 16.5 °C; sunshine, 7.75 h; rainfall, 3.75 mm and windspeed, 2.96 km/h. This experiment had a completely randomized design with three replicates. Each plastic cup was filled with 0.4 kg sandy loamy soil, and 5 seeds of broccoli were sown in each pot. Seeds were germinated after one week and after 7 days of germination, thinning was done to maintain three seedlings in each pot. After thinning, maintained three plants per plastic cup and drought stress (60% FC) was initiated on the basis of soil field capacity in addition to control (100% FC). The required field capacity for drought stressed plants maintained after ten days. The sandy loam soil having sand 60%, silt 29.5%, clay 10.5%, and saturation percentage 32%. Nitric oxide (NO; 0.02 mM) was applied as a presowing as well as foliar spray. For pre-sowing application, broccoli seeds were soaked for fifteen hours in nitric oxide (NO) solution (0.02 mM) by dissolving 0.02 g sodium nitroprusside (SNP) as a NO donor in 500 ml of distilled water. After 21 days of water stress treatment, foliar application, 0.02 mM NO was applied as a foliar spray on broccoli plants. NO (0.02 mM) solution was prepared by mixing SNP with 500 ml distilled water and 0.1% of Tween-20 as a surfactant. Before foliar application, soil was covered with a polythene sheet and then NO solution sprayed to each leaf with the help of a hand sprayer. After 12 days of NO application, one plant was collected for recording growth parameters and leaves of remaining two plants were collected and stored at -20 °C for different analyses.
2.5. Total phenolics The filtrate (0.1 ml, same as used for chlorophyll), distilled water (2 ml) and Folin-Ciocalteuʼs phenol reagent (1 ml) were taken in test tubes and were shaken well. After that, 5 ml of Na2CO3 (20%) were added to each tube and made the volume 10 ml by adding distilled water. Then, the OD was read at 750 nm using a spectrophotometer following Julkenen-Titto (1985). 2.6. Ascorbic acid (AsA) It was recorded following Mukherjee and Choudhuri (1983). Leaf sample (0.5 g) was triturated in 10 ml TCA (6%) and centrifuged at 10,000 rpm for about 10 min. The supernatant (4 ml) from each sample was taken into labeled test tubes and added 2 ml of 2% 2, 4 - DNPH (2 g in 100 ml 9 N H2SO4) followed by 1 drop of thiourea (10%, prepared in 70% ethanol) into it. The samples were placed in a water bath for 15 min and then cooled. Later on, 5 ml H2SO4 (80%) were added to each tube and OD read at 530 nm. 2.7. Hydrogen peroxide (H2O2) It was estimated according to the method of Velikova et al. (2000). The supernatant (0.5 ml, same as used for AsA) was taken in test tube and added 0.5 ml potassium phosphate buffer (pH 7) and 1 ml potassium iodide (1 M) to it. The mixture was vortexed and noted the absorbance at 390 nm. 2.8. Antioxidants enzymes
2.1. Growth parameters Leaf sample (0.5 g) was ground in 5 ml potassium phosphate buffer (7.8 pH) in ice, centrifuged at 10,000 rpm and used the supernatant to determine total soluble proteins and activities of catalase (CAT),
Fresh and dry weights of shoot were recorded using an electronic balance and lengths of shoots were measured using meter rule. 8
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drought stress (Table 1). It was observed that foliar application of NO was more effective than pre-sowing of NO under both well-watered and drought stress (Figs. 1, 2). Proline content of broccoli plants showed no response to water deficit conditions. Similarly, exogenous application of NO had nonsignificant effect on proline contents of broccoli plants under drought stress (Table 1). Water shortage significantly (P ≤ 0.05) decreased the glycine betaine (GB) contents of broccoli plants while exogenously applied NO significantly (P ≤ 0.05) enhanced the GB content of broccoli plants under water shortage (Table 1). It was noticed that foliar fed plants showed more improvement in GB contents than did the pre-sowing plants (Fig. 2). Water deficiency notably (P ≤ 0.001) increased the ascorbic acid (AsA) and total total phenolics content of broccoli plants. Exogenous application of NO was effective as pre-sowing of NO on AsA contents and both modes (pre-sowing and foliar) for total phenolics of broccoli plants under drought stress (Fig. 2). Water deficit conditions significantly increased H2O2 (P ≤ 0.01) contents of broccoli plants (Table 1). However, exogenously applied NO had no marked effect on H2O2 contents of broccoli plants under water deficit conditions (Table 1). Water deficiency and exogenous application of NO had no significant effect on total soluble proteins of broccoli plants (Table 1; Fig. 2). A significant increase was observed in the activity of SOD enzyme (P ≤ 0.001) of broccoli plants under water limited conditions (Table 1). Exogenous application of NO significantly affected the activity of SOD enzyme (P ≤ 0.05) of broccoli plants under water deficit conditions (Table 1). Foliar-applied NO had more prominent effect on broccoli plants as compared to pre-sowing seed treatment of NO under control and water deficiency (Fig. 3). Drought stress had no marked effect on the activity of POD enzyme in broccoli plants (Table 1). However, exogenously applied NO significantly affected he activity of POD (P ≤ 0.05) of broccoli plants under drought stress (Table 1). Foliar application of NO had more pronounced effects on broccoli plants as compared to the pre-sowing of NO under well-watered and water deficit conditions (Fig. 3). Water shortage and exogenous application of NO slightly increased the activity of CAT enzyme in both stressed and unstressed broccoli
peroxidase (POD) and superoxide dismutase (SOD). The activities of CAT and POD enzymes were determined using the protocol of Chance and Maehly (1955) while SOD according to Van Rossum et al. (1997). For CAT activity, the supernatant (0.1 ml) was treated with 1 ml H2O2 (5.9 mM) and 2.8 ml potassium phosphate buffer (7.8 pH; 50 mM) in quartz cuvette and read the absorbance at 240 nm after every 20 s till 180 s with the help of a spectrophotometer. For POD activity, 50 μl supernatant, 750 μl potassium phosphate buffer (pH 7.8, 50 mM) followed by100 μl guiacol (20 mM) and 100 μl H2O2 (40 mM) were added in a cuvette and read the absorbance at 470 nm after every 20 s up to 120 s on a spectrophotometer. For SOD activity, to an aliquot of 50 μl of the supernatant, 400 μl distilled water, 50 μl NBT, 50 μl riboflavin (0.026 g in 30 ml buffer having pH 7.8), 250 μl potassium phosphate buffer (pH 7.8, 50 mM), 100 μl methionine (0.44 g in 30 ml buffer having pH 7.8) and 100 μl Triton-X (75 μl in 30 ml buffer solution) were added sequentially in a plastic cuvette, placed all cuvettes containing samples under light for 15 min and read the absorbance at 560 nm using a spectrophotometer. 2.9. Statistical analysis A two-factor ANOVA was worked from the data of each attribute using the statistical software Cohort Costat program. Mean data were compared using least significance difference at 0.05% probability level. 3. Results Drought stress (60% FC) significantly reduced the shoot fresh and dry weights (P ≤ 0.01, 0.001) and shoot length (P ≤ 0.001) of broccoli (Brassica oleracea L.) plants (Table 1). However, exogenously applied nitric oxide (0.2 mM NO) considerably enhanced the shoot fresh and dry weights (P ≤ 0.01, P ≤ 0.001), and shoot length (P ≤ 0.001) of broccoli plants under drought conditions (Table 1). Foliar-applied NO showed more pronounced results as compared to pre-sowing application of NO on broccoli plants under both control and water deficit conditions (Fig. 1). Water deficit conditions markedly reduced chlorophyll a (P ≤ 0.001) and b (P ≤ 0.01) contents of broccoli plants (Table 1). While exogenous application of NO improved significantly chlorophyll a (P ≤ 0.001), and chlorophyll b (P ≤ 0.05) contents of broccoli plants under
Table 1 Mean squares from two way analysis of variance of different morphological and biochemical parameters of 72 days old broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under well watered (control, 100% FC) and water deficit (60% FC) conditions. Source of variations
df
Shoot FW
Shoot DW
Shoot length
Chlorophyll a
Drought stress (D) Nitric oxide (NO) D × NO Error
1 2 2 18
Drought (D) Nitric oxide (NO) D × NO Error
1 2 2 18
Drought (D) Nitric oxide (NO) D × NO Error
1 2 2 18
0.0415*** 0.0148*** 0.0055* 0.0013 AsA 158.4*** 19.7082ns 2.7543ns 6.8216 SOD 248.2*** 71.61* 1.588ns 15.63
1 2 2 18
0.1962*** 0.1109*** 0.0298* 0.0075 Proline 1.5084ns 4.4056ns 1.7277ns 1.8546 H2O2 515606.8** 83889.4ns 16534.1ns 44461.9 CAT 0.3609* 0.0745ns 0.0093ns 0.123
50.750*** 21.0004*** 2.3654ns 1.7331 GB 140.373* 152.395* 4.8300ns 25.5433 TSP 0.2571ns 1.2262* 0.0071ns 0.3254
Drought (D) Nitric oxide (NO) D × NO Error
2.6867** 2.1362** 0.1260ns 0.2235 Chlorophyll b 0.8050** 0.3195* 0.0461ns 0.0838 Total Phenolics 92.82ns 118.325* 1.5275ns 24.74 POD 0.0016ns 0.0067*** 0.0017* 4.9769
ns=non-significant; *, ** and *** = significant at 0.05, 0.01 and 0.001 levels, respectively. DW, Dry weight; FW, Fresh weight; GB, Glycinebetaine; AsA, Ascorbic acid; H2O2, Hydrogen peroxide; TSP, Total soluble proteins; SOD, Superoxide dismutase; POD, Peroxidase; CAT, Catalase. 9
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Fig. 1. Fresh and dry weights and lengths of shoot, chlorophyll a and b contents of broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under control and drought.
acids in plant cell metabolism (Souri and Hatamian, 2019). It is a fundamental and basic fact in plant science that chlorophyll molecules are essential for photosynthesis, but water shortage can destroy chlorophyll molecules and inhibit their synthesis, which in turn reduces photosynthesis rate in plants (Heba and Samia, 2014). In our study, water shortage decreased the chlorophyll contents of broccoli plants, analogous to what has been observed in soybean (Heba and Samia, 2014), wheat (Raza et al., 2016), radish (Akram et al., 2016) and cucumber (Naz et al., 2016) plants. They observed that drought stress decreased the chlorophyll contents of plants which thereby reduced the photosynthetic rate. However, exogenous application of NO significantly improved chlorophyll contents of broccoli plants under water shortage. Similar findings were observed in cucumber (Fan et al., 2007), rice (Farooq et al., 2009), Malus hupehensis (Cao et al., 2011), Malus rootstocks (Zhang et al., 2016). It has been reported that NO can enhance the synthesis complexes and protein molecules of chloroplasts and mitochondria (Simontacchi et al., 2015). It is now well evident that glycine betaine (GB) accumulates in plants under water shortage and is involved in improving the defensive antioxidant system by scavenging the reactive oxygen species (ROS)
plants (Table 1).
4. Discussion This study was carried out to assess whether or not and which mode of exogenously applied nitric oxide (0.02 mM) might improve oxidative defense in broccoli (Brassica oleracea L.) plants under drought stress. In the current study, water deficiency reduced the dry and fresh weight of shoots and shoot length of broccoli plants which is similar to the findings already reported in cauliflower (Latif et al., 2016; Mukhtar et al., 2016), cucumber (Naz et al., 2016) and carrot (Razzaq et al., 2017). The drought induced reduction in all these crops was ascribed to lipid peroxidation, alteration in hormone levels and nutrition uptake. However, in the current study exogenously applied nitric oxide enhanced shoot fresh and dry weights and shoot length of broccoli plants. It is similar to what has earlier been observed in wheat (Tian and Lei, 2006) and cucumber (Arasimowicz-Jelonek et al., 2009). NO is believed to enhance vegetative growth and root morphology of plants (Shao et al., 2010). Many studies show the semi hormonal effects of various nitrogenous compounds including nitrate, ammonium and some amino 10
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Fig. 2. Leaf proline, glycine betaine (GB), ascorbic acid (AsA), total phenolics, hydrogen peroxide and total soluble protein (TSP) contents of broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under control and drought stress (Mean ± S.E.) Letters (A–C) showing least significance difference among mean values.
deficit conditions was observed. Our results are also in contrast to those in quinoa (Aziz et al., 2018) wherein an increase in total phenolics under drought conditions was observed. However, exogenous application of NO enhanced the total phenolics of broccoli plants under drought stress. The increase in the total phenolics may be attributed to the fact that NO acts as a signaling molecule to activate the antioxidant defense system (Simontacchi et al., 2015). Plants possess enzymatic and non-enzymatic antioxidative defense system to protect themselves from stresses including that caused by water deficit conditions (Akram et al., 2017). In our study, drought stress increased the activity of SOD and CAT, but had no effect on POD enzyme of broccoli plants under water deficiency. Our findings for the SOD enzyme are similar but for the POD are in contrast to those in corn (Darvishan et al., 2013), wheat (Selote and Khanna-Chopra, 2010), and quinoa (Aziz et al., 2018), wherein enhanced activities of SOD and POD have been observed under water scarce regimes. On the other hand, exogenous application of NO improved the activities of SOD and POD of broccoli plants under water deficiency. This is similar to what has been found in turfgrasses (Boogar et al., 2014), hulless barley (Gan et al., 2015) and Malus rootstocks (Zhang et al., 2016). The increase in
(Ashraf et al., 2011; Vijayalakshmi et al., 2016). In the present study, water deficit conditions decreased the glycine betaine (GB) contents of broccoli plants, which is contrary to what has been observed in wheat (Singh and Bhardwaj, 2016) and quinoa (Aziz et al., 2018), wherein enhanced levels of GB have been found under water deficit conditions. Nonetheless, exogenously applied NO enhanced the GB content of broccoli plants under water shortage, analogous to what has been found in maize (Zhang et al., 2012). Zhang et al. (2012) have reported that exogenous application of NO enhanced the content and activity of choline and betaine aldehyde dehydrogenase, which are involved in GB synthesis (Sithtisarn et al., 2007). In addition, it has been shown that exogenous application of glycine amino acid alone or in form of chelated with nutrients can enhance plant growth particularly under adverse climatic conditions (Souri and Hatamian, 2019, 2019). Under oxidative stress induced by drought stress, phenolic compounds are accumulated, which can safeguard fatty acids (Amri et al., 2017). In this study, water deficit conditions did not affect the total phenolic content in broccoli plants. Our findings are in contrast to those reported in maize (Moharramnejad et al., 2015) and canola (Dawood and Sadak, 2014) wherein a decrease in total phenolics under water 11
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Fig. 3. Activities of superoxide dismutase (SOD), catalse (CAT) and peroxidase (POD) enzymes of broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under control and drought stress (Mean ± S.E.), letters (A-C) showing least significance difference among mean values.
antioxidant activities could be due to the role of NO in increasing the capability of cellular antioxidative defense system (Santa-Cruz et al., 2010). Overall, water deficit conditions decreased the growth, glycine betaine, chlorophyll contents, while it enhanced ascorbic acid (AsA), hydrogen peroxide and SOD and CAT enzymes activities. Drought stress did not affect the contents of proline, total phenolics, total soluble proteins as well as the activities of POD enzyme. Exogenously applied NO enhanced fresh and dry weights of shoots, shoot length, chlorophyll contents, GB, total phenolics, total soluble proteins and the activities of CAT, SOD and POD in broccoli plants under water deficiency. However, exogenous application of NO had no effect on free proline content, AsA and H2O2 of broccoli plants under water deficit conditions. It was also observed that foliar application of NO was more effective in enhancing the drought tolerance in broccoli plants as compared to pre-sowing application of NO. Therefore, foliar application of NO could be beneficial in enhancing the oxidative defense system of broccoli plants under water deficit conditions.
249–296. Asrar, A.W.A., Elhindi, K.M., 2011. Alleviation of drought stress of marigold (Tagetes erecta) plants by using arbuscular mychorrhizal fungi. Saudi J. Biol. Sci. 18, 93–98. Aziz, A., Akram, N.A., Ashraf, M., 2018. Influence of natural and synthetic vitamin C (ascorbic acid) on primary and secondary metabolites and associated metabolism in quinoa (Chenopodium quinoa Willd.) plants under water deficit regimes. Plant Physiol. Biochem. 123, 192–203. Bates, L.S., Waldren, R.P., Teare, L.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Boogar, A.R., Salehi, H., Jowkar, A., 2014. Exogenous nitric oxide alleviates oxidative damage in turfgrasses under drought stress. S. Afr. J. Bot. 92, 78–82. Buet, A., Simontacchi, M., 2015. Nitric oxide and plant iron homeostasis. Ann. New York Acad. Sci. 1340, 39–46. Cao, H., Wang, X.W., Zou, Y.M., Shu, H.R., 2011. Effects of exogenous nitric oxide on chlorophyll fluorescence parameters and photosynthesis rate in Malus hupehensis seedlings under water stress. Acta Hort. Sin. 38, 613–620. Chance, B., Maehly, A., 1955. Assay of catalase and peroxidase. Meth. Enzymol. 2, 764–817. Chen, K., Chen, L., Fan, J., Fu, J., 2013. Alleviation of heat damage to photosystem II by nitric oxide in tall fescue. Photosyn. Res. 116, 21–31. Darvishan, M., Tohidi-Moghadam, H.R., Zahedi, H., 2013. The effect of foliar application of ascorbic acid (vitamin C) on physiological and biochemical changes of corn (Zea mays L.) under irrigation withholding in different growth stages. Maydica 58, 195–200. Dawood, M.G., Sadak, M.S., 2014. Physiological role of glycine betaine in alleviating the deleterious effects of drought stress on canola plants (Brassica napus L.). Mid. East J. Agric. Res. 3, 943–954. De Figueiredo, S.M., Binda, N.S., Nogueira-Machado, J.A., Vieira-Filho, S.A., Caligiorne, R.B., 2015. The antioxidant properties of organo-sulfur compounds (sulforaphane). Rec. Pat. Endocr. Metab Immune Drug Discov. 9, 24–33. Fan, H., Guo, S., Jiao, Y., Zhang, R., Li, J., 2007. Effects of exogenous nitric oxide on growth, active oxygen species metabolism, and photosynthetic characteristics in cucumber seedlings under NaCl stress. Front. Agric. China 1, 308–314. Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., Basra, S.M.A., 2009. Plant drought stress effects, mechanisms and management. Agron. Sustain. Develop. 29, 185–212. Fernández-Marcos, M., Sanz, L., Lewis, D.R., Muday, G.K., Lorenzo, O., 2011. Nitric oxide causes root apical meristem defects and growth inhibition while reducing PINFORMED 1 (PIN1)-dependent acropetal auxin transport. Proc. Nat. Acad. Sci. 108, 18506–18511. Galahitigama, G.A.H., Wathugala, D.L., 2016. Pre-sowing seed treatments improves the growth and drought tolerance of rice (Oryza sativa L.). Imp. J. Int. Res. 2, 1074. Gan, L., Wu, X., Zhong, Y., 2015. Exogenously applied nitric oxide enhances the drought tolerance in hulless barley. Plant Prod. Sci. 18, 52–56. Grieve, C.M., Grattan, S.R., 1983. Rapid assay for the determination of water soluble quaternary ammonium compounds. Plant Soil 70, 303–307.
References Akram, N.A., Waseem, M., Ameen, R., Ashraf, M., 2016. Trehalose pretreatment induces drought tolerance in radish (Raphanus sativus L.) plants: some key physio-biochemical traits. Acta Physiol. Plant. 38, 3. Akram, N.A., Shafiq, F., Ashraf, M., 2017. Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front. Plant Sci. 8, 613. Akram, N.A., Shafiq, F., Ashraf, M., 2018. Peanut (Arachis hypogaea L.): a prospective legume crop to offer multiple health benefits under changing climate. Compr. Rev. Food Sci. Food Saf. 17 (5), 1325–1338. Amri, Z., Lazreg-Aref, H., Mekni, M., El-Gharbi, S., Dabbaghi, O., Mechri, B., Hammami, M., 2017. Oil characterization and lipids class composition of pomegranate seeds. Biol. Med. Res. Int Article No. 8. Arasimowicz-Jelonek, M., Floryszak-Wieczorek, J., Kubiś, J., 2009. Involvement of nitric oxide in water stress-induced responses of cucumber roots. Plant Sci. 177, 682–690. Arnon, D.T., 1949. Copper enzyme in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Ashraf, M., Akram, N.A., Al-Qurainy, F., Foolad, M.R., 2011. Drought tolerance: roles of organic osmolytes, growth regulators, and mineral nutrients. Adv. Agron. 111,
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A. Munawar, et al.
defense system in carrot (Daucus carota L.) plants. Sci. Hort. 225, 373–379. Santa-Cruz, D.M., Pacienza, N.A., Polizio, A.H., Balestrasse, K.B., Tomaro, M.L., Yannarelli, G.G., 2010. Nitric oxide synthase-like dependent NO production enhances heme oxygenase up-regulation in ultraviolet-B-irradiated soybean plants. Phytochemistry 71, 1700–1707. Sanz, L., Albertos, P., Mateos, I., Sánchez-Vicente, I., Lechón, T., Fernández-Marcos, M., Lorenzo, O., 2015. Nitric oxide (NO) and phytohormones crosstalk during early plant development. J. Exp. Bot. 66, 2857–2868. Selote, D.S., Khanna-Chopra, R., 2010. Antioxidant response of wheat roots to drought acclimation. Protoplasma 245, 153–163. Shao, Y., Zhang, S., Engelhard, M.H., Li, G., Shao, G., Wang, Y., Liu, J., Aksay, I.A., Lin, Y., 2010. Nitrogen-doped graphene and its electrochemical applications. J. Mater. Chem. 20, 7491–7496. Simontacchi, M., Galatro, A., Ramos-Artuso, F., Santa-María, G.E., 2015. Plant survival in a changing environment: the role of nitric oxide in plant responses to abiotic stress. Front. Plant Sci. 6, 977. Singh, N., Bhardwaj, R.D., 2016. Ascorbic acid alleviates water deficit induced growth inhibition in wheat seedlings by modulating levels of endogenous antioxidants. Biologia 71, 402–413. Sithtisarn, S., Harinasut, P., Pornbunlualap, S., Cha-um, S., 2007. Accumulation of Glycinebetaine and Betaine Aldehyde Dehydrogenase Activity in Eucalyptus camaldulensis Clone T5 Under in Vitro Salt Stress. Doctoral dissertation. Kasetsart University. Sotelo, T., Soengas, P., Velasco, P., Rodríguez, V.M., Cartea, M.E., 2014. Identification of metabolic QTLs and candidate genes for glucosinolate synthesis in Brassica oleracea leaves, seeds and flower buds. PLoS One 9, 91428. Souri, M.K., Hatamian, M., 2019. Aminochelates in plant nutrition; a review. J. Plant Nutr. 42, 67–78. Tian, X., Lei, Y., 2006. Nitric oxide treatment alleviates drought stress in wheat seedlings. Biol. Plant. 50, 775–778. Van Rossum, M.W., Alberda, M., Van der Plas, L.H., 1997. Role of oxidative damage in tulip bulb scale micropropagation. Plant Sci. 130, 207–216. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Sci. 151, 59–66. Vijayalakshmi, T., Vijayakumar, A.S., Kiranmai, K., Nareshkumar, A., Sudhakar, C., 2016. Salt stress induced modulations in growth, compatible solutes and antioxidant enzymes response in two cultivars of safflower (Carthamustinctorius L. cultivar TSF1 and cultivar SM) differing in salt tolerance. Am. J. Plant Sci. 7, 1802. Wani, S.H., Kumar, V., Shriram, V., Sah, S.K., 2016. Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J. 4, 162–176. Yuan, G.F., Sun, B., Yuan, J., Wang, Q.M., 2009. Effects of different cooking methods on health promoting compounds of broccoli. J. Zhejiang Univ. Sci. B 10, 580–588. Zhang, L.X., Zhai, Y.Y., Li, Y., Zhao, Y.O., Lv, L.X., Gao, M., Liu, J.C., Hu, J.J., 2012. Effects of nitrogen forms and drought stress on growth, photosynthesis and some physico-chemical properties of stem juice of two maize cultivars (Zea mays L.) at elongation stage. Pak. J. Bot. 44, 1405–1412. Zhang, L., Li, X., Li, X., Wei, Z., Han, M., Zhang, L., Li, B., 2016. Exogenous nitric oxide protects against drought-induced oxidative stress in Malus rootstocks. Turk. J. Bot. 40, 17–27.
Hatamian, M., Nejad, A.R., Kafi, M., Souri, M.K., Shahbazi, K., 2018. Interactions of lead and nitrate on growth characteristics of ornamental judas tree (Cercis siliquastrum). Open Agric. 3, 386–392. Heba, I.M., Samia, A.A., 2014. Influence of garlic extract on enzymatic and non-enzymatic antioxidants in soybean plants (Glycine max L.) grown under drought stress. J. Life Sci. 11, 47–58. Hichri, I., Boscari, A., Castella, C., Rovere, M., Puppo, A., Brouquisse, R., 2015. Nitric oxide: a multifaceted regulator of the nitrogen-fixing symbiosis. J. Exp. Bot. 66, 2877–2887. Jeffery, E.H., Araya, M., 2009. Physiological effects of broccoli consumption. Phytochem. Rev. 8, 283–298. Julkenen-Titto, R., 1985. Phenolic constituents in the leaves of northern willows: Methods for the analysis of certain phenolics. J. Agric. Food Chem. 33, 213–217. Khan, M.A.M., Ulrichs, C., Mewis, I., 2010. Effects of water stress and aphid herbivory flavonoids in broccoli (Brassica oleracea var. italic Plenck). J. Appl. Bot. Food Qual. 84, 178–182. Krasuska, U., Ciacka, K., Andryka-Dudek, P., Bogatek, R., Gniazdowska, A., 2015. “Nitrosative Door” in seed dormancy alleviation and germination. React. Oxygen Nitrogen Species Signal. Commun. Plants 215–237. Latif, M., Akram, N.A., Ashraf, M., 2016. Regulation of some biochemical attributes in drought-stressed cauliflower (Brassica oleracea L.) by seed pre-treatment with ascorbic acid. J. Hort. Sci. Biotechnol. 91, 129–137. Liu, F., Guo, F.Q., 2013. Nitric oxide deficiency accelerates chlorophyll breakdown and stability loss of thylakoid membranes during dark-induced leaf senescence in Arabidopsis. PLoS One 8, e56345. Lombardo, M.C., Graziano, M., Polacco, J.C., Lamattina, L., 2006. Nitric oxide functions as a positive regulator of root hair development. Plant Signal. Behav. 1, 28–33. López-Berenguer, C., García-Viguera, C., Carvajal, M., 2006. Are root hydraulic conductivity responses to salinity controlled by aquaporins in broccoli plants? Plant Soil 279, 13–23. Lozano-Juste, J., León, J., 2011. Nitric oxide regulates DELLA content and PIF expression to promote photomorphogenesis in Arabidopsis. Plant Physiol. 156, 1410–1423. Moharramnejad, S., Sofalian, O., Valizadeh, M., Asgari, A., Shiri, M., 2015. Proline, glycine betaine, total phenolics and pigment contents in response to osmotic stress in maize seedlings. J. Biosci. Biotechnol. 4. Mukherjee, S.P., Choudhuri, M.A., 1983. Implication of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol. Plant. 58, 166–170. Mukhtar, A., Akram, N.A., Aisha, R., Shafiq, S., Ashraf, M., 2016. Foliar-applied ascorbic acid enhances antioxidative potential and drought tolerance in cauliflower (Brassica oleracea L. var. Botrytis). Agrochimica 60, 107–113. Naz, H., Akram, N.A., Ashraf, M., 2016. Impact of ascorbic acid on growth and some physiological attributes of cucumber (Cucumis sativus) plants under water-deficit conditions. Pak. J. Bot. 48, 877–883. Ravikumar, C., 2015. Therapeutic potential of Brassica oleracea (broccoli)-a review. Int. J. Drug Dev. Res. 7, 9–10. Raza, M.A.S., Saleem, M.F., Khan, I.H., Shah, G.M., Raza, A., 2016. Bio-economics of foliar applied GB and K on drought stressed wheat (Triticum aestivum L.). ARPN J. Agric. Biol Sci. 11, 1. Razzaq, M., Akram, N.A., Ashraf, M., Naz, H., Al-Qurainy, F., 2017. Interactive effect of drought and nitrogen on growth, some key physiological attributes and oxidative
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Nitric oxide regulates oxidative defense system, key metabolites and growth of broccoli (Brassica oleracea L.) plants under water limited conditions
T
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Aneeqa Munawara, Nudrat Aisha Akrama, , Abrar Ahmada, Muhammad Ashrafb a b
Department of Botany, Government College University, Faisalabad, Pakistan University of Agriculture Faisalabad, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: Broccoli (Brassica oleracea) Drought stress Nitric oxide Antioxidants
Nitric oxide (NO) is a diffusible gaseous molecule and has been under wide consideration because of its ability to mitigate adverse effects of several abiotic stresses on plants. In the current study, it was determined whether or not exogenous application (presowing seed treatment and foliar application) of sodium nitroprusside (SNP), donor of nitric oxide (NO), could alleviate the drastic effects of drought stress on broccoli plants. The broccoli seeds were soaked in 0.02 mM NO solution or distilled water for pre-sowing and control treatments, respectively. Two levels of water stress (control, 100% field capacity (FC) and 60% FC) were applied to 4 week-old broccoli (Brassica oleracea L.) plants. Foliar treatment of NO (0.02 mM) was applied to broccoli plants after 3 weeks of initiation of drought stress. After 12 days of foliar application, leaf samples were collected to determine photosynthetic and antioxidant activities as well as other biochemical parameters. The results showed that water deficit conditions decreased the shoot fresh and dry weights and shoot length, glycine betaine, and chlorophyll contents, while it enhanced ascorbic acid (AsA), hydrogen peroxide and activities of CAT and SOD enzymes. However, exogenously applied NO as a presowing seed treatment or foliar spray enhanced the fresh and dry biomass of shoot, shoot length, chlorophyll contents, GB, total phenolics, total soluble proteins and activities of SOD and POD enzymes in broccoli plants under water deficiency. It was also observed that foliar application of NO was more effective in enhancing the drought tolerance in broccoli plants as compared to pre-sowing application of NO. Therefore, foliar as well as pre-sowing application of NO could be helpful in up-regulating the oxidative defense system of broccoli plants under water deficit conditions.
1. Introduction Plant growth restriction is a ubiquitous phenomenon in agricultural food production in many parts of the world, mainly due to various environmental stresses (Hatamian et al., 2018; Souri and Hatamian, 2019). Abiotic stress such as drought stress has drastic effects on the development, growth and yield of plants (Wani et al., 2016). Khan et al. (2010) reported that water deficit stress reduces the mean productivity of grain crops from 17 to 70%. Drought stress reduces plant water content, nutrient uptake, leaf size, stem elongation, root proliferation, gas exchange, water use efficiency, transpiration rate, germination rate, cell division and expansion (Farooq et al., 2009; Galahitigama and Wathugala, 2016). Drought stress impairs the enzyme activity and decreases the energy supply (Farooq et al., 2009). Drought stress results into impaired stomatal conductance, turgor loss and nutritional imbalance (Galahitigama and Wathugala, 2016). Drought stress decreases the seed production by plants as it severely affects pollination (Asrar
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and Elhindi, 2011). Water deficit also causes oxidative stress which damages the cellular membranes and impairs photosynthesis (Aziz et al., 2018). Many studies have used various organic or mineral compounds to alleviate adverse effects of abiotic stresses on plant production (Souri and Hatamian, 2019). To alleviate the adverse effects of drought stress on plants, a number of means are in practice these days. Of these, exogenous application of a variety of plant growth regulators (PGRs) are being used to improve stress tolerance in plants (Akram et al., 2018). Different nitrogen compounds including various amino acids, peptides and nitric oxide have been shown to reduce the adverse effects of abiotic stresses on plants (Souri and Hatamian, 2019). Recently, it has been shown that ornamental judas tree (Cercis siliquastrum) have better tolerance to lead and cadmium toxicity under higher nitrate application in irrigation water (Hatamian et al., 2018). Nitric oxide (NO) is contemplated as one of the potential PGRs which can effectively offset the adverse effects of different abiotic stresses including drought
Corresponding author. E-mail address: [email protected] (N.A. Akram).
https://doi.org/10.1016/j.scienta.2019.04.072 Received 3 January 2019; Received in revised form 25 April 2019; Accepted 26 April 2019 Available online 03 May 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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2.2. Chlorophyll contents
stress. Nitric oxide is a gas which acts as a signaling molecule in plants under abiotic stress conditions (Boogar et al., 2014). Nitric oxide is known to break seed dormancy and promote germination (Krasuska et al., 2015) as well as it stimulates growth of root hairs (Lombardo et al., 2006), lateral and adventitious roots (Fernández-Marcos et al., 2011). Several reports have shown that NO is involved in the maintenance of chlorophyll contents (Liu and Guo, 2013), stomatal movements (Chen et al., 2013), vegetative growth (Lozano-Juste and León, 2011), homeostasis of iron (Buet and Simontacchi, 2015), production of nodules by symbiosis (Hichri et al., 2015), and hormonal metabolism (Sanz et al., 2015). Nitric oxide can also scavenge ROS and calcium signaling thereby improving the immunity of plants (Simontacchi et al., 2015). It has been reported that like other chemicals, sodium nitroprusside as a donor of NO is being used exogenously to mitigate the adverse effects of different abiotic stresses including drought stress (Boogar et al., 2014). Broccoli (Brassica oleracea L.) is considered as one of the major vegetables (López-Berenguer et al., 2006) and is cultivated throughout the world (Sotelo et al., 2014). It has high contents of minerals and vitamins and has beneficial effects on the health of human beings (Jeffery and Araya, 2009). Broccoli has anti-cancerous properties (De Figueiredo et al., 2015) as it is a good source of glycosinolates, fibers and minerals such as Fe, K, etc, vitamin C and K (Yuan et al., 2009; Ravikumar, 2015). The main objective of this study was to determine whether or not exogenously applied sodium nitroprusside, donor of NO, as a presowing seed treatment or foliar application could mitigate the adverse effects of drought stress on broccoli plants particularly with relation to the oxidative defense system.
Chlorophyll contents were measured using the method of Arnon (1949). Fresh leaf sample (0.5 g) was dipped into 10 ml acetone (80%) and allowed it to stand for one night in a cool place. Chlorophyll a and b contents were determined using a spectrophotometer. 2.3. Leaf free proline It was determined according to the method of Bates et al. (1973). Leaf samples (0.5 g) were ground in 10 ml of sulfosalicylic acid (3%) solution and filtered. The filtrate (2 ml) of each sample was taken into a separate labeled test tubes, and added acidic ninhydrin (1.26 g ninhydrin + 30 ml glacial acetic acid + 20 ml 6 M ortho-phosphoric acid) solution (2 ml) and glacial acetic acid (2 ml). Later on, the mixture incubated at 100 °C for 60 min. After that, toluene (4 ml) was added to each test tube and all tubes were shaken well. The absorbance of the treated samples was read at 520 nm using a spectrophotometer. 2.4. Glycine betaine (GB) GB in leaf tissue was determined following the Grieve and Grattan (1983) method. Leaf sample (0.5 g) was extracted in 10 ml distilled water and allowed it to stay at 4 °C for one night. After that, these samples were centrifuged at 10,000 rpm for 10 min. The supernatant (1 ml) from each sample was taken in a separate labeled test tube and added 1 ml of H2SO4 (2N) followed by 0.2 ml KI3 (1 M). All these test tubes were placed in an ice bath and allowed them to cool for one and half hour. Later on, 2.8 ml distilled water followed by 1–2 dichloroethane (6 ml) were added to them. Two layers were formed, discarded the upper layer and used the lower layer for taking readings at 365 nm using a spectrophotometer.
2. Materials and methods This study was conducted to evaluate the role of different modes (pre-sowing and foliar) of exogenously applied nitric oxide (NO) in improving drought tolerance of broccoli (Brassica oleracea L.). An experiment was conducted in the research area of GC University Faisalabad, Pakistan during November-January. During this period, average climate conditions were recorded as: temperature, 16.5 °C; sunshine, 7.75 h; rainfall, 3.75 mm and windspeed, 2.96 km/h. This experiment had a completely randomized design with three replicates. Each plastic cup was filled with 0.4 kg sandy loamy soil, and 5 seeds of broccoli were sown in each pot. Seeds were germinated after one week and after 7 days of germination, thinning was done to maintain three seedlings in each pot. After thinning, maintained three plants per plastic cup and drought stress (60% FC) was initiated on the basis of soil field capacity in addition to control (100% FC). The required field capacity for drought stressed plants maintained after ten days. The sandy loam soil having sand 60%, silt 29.5%, clay 10.5%, and saturation percentage 32%. Nitric oxide (NO; 0.02 mM) was applied as a presowing as well as foliar spray. For pre-sowing application, broccoli seeds were soaked for fifteen hours in nitric oxide (NO) solution (0.02 mM) by dissolving 0.02 g sodium nitroprusside (SNP) as a NO donor in 500 ml of distilled water. After 21 days of water stress treatment, foliar application, 0.02 mM NO was applied as a foliar spray on broccoli plants. NO (0.02 mM) solution was prepared by mixing SNP with 500 ml distilled water and 0.1% of Tween-20 as a surfactant. Before foliar application, soil was covered with a polythene sheet and then NO solution sprayed to each leaf with the help of a hand sprayer. After 12 days of NO application, one plant was collected for recording growth parameters and leaves of remaining two plants were collected and stored at -20 °C for different analyses.
2.5. Total phenolics The filtrate (0.1 ml, same as used for chlorophyll), distilled water (2 ml) and Folin-Ciocalteuʼs phenol reagent (1 ml) were taken in test tubes and were shaken well. After that, 5 ml of Na2CO3 (20%) were added to each tube and made the volume 10 ml by adding distilled water. Then, the OD was read at 750 nm using a spectrophotometer following Julkenen-Titto (1985). 2.6. Ascorbic acid (AsA) It was recorded following Mukherjee and Choudhuri (1983). Leaf sample (0.5 g) was triturated in 10 ml TCA (6%) and centrifuged at 10,000 rpm for about 10 min. The supernatant (4 ml) from each sample was taken into labeled test tubes and added 2 ml of 2% 2, 4 - DNPH (2 g in 100 ml 9 N H2SO4) followed by 1 drop of thiourea (10%, prepared in 70% ethanol) into it. The samples were placed in a water bath for 15 min and then cooled. Later on, 5 ml H2SO4 (80%) were added to each tube and OD read at 530 nm. 2.7. Hydrogen peroxide (H2O2) It was estimated according to the method of Velikova et al. (2000). The supernatant (0.5 ml, same as used for AsA) was taken in test tube and added 0.5 ml potassium phosphate buffer (pH 7) and 1 ml potassium iodide (1 M) to it. The mixture was vortexed and noted the absorbance at 390 nm. 2.8. Antioxidants enzymes
2.1. Growth parameters Leaf sample (0.5 g) was ground in 5 ml potassium phosphate buffer (7.8 pH) in ice, centrifuged at 10,000 rpm and used the supernatant to determine total soluble proteins and activities of catalase (CAT),
Fresh and dry weights of shoot were recorded using an electronic balance and lengths of shoots were measured using meter rule. 8
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drought stress (Table 1). It was observed that foliar application of NO was more effective than pre-sowing of NO under both well-watered and drought stress (Figs. 1, 2). Proline content of broccoli plants showed no response to water deficit conditions. Similarly, exogenous application of NO had nonsignificant effect on proline contents of broccoli plants under drought stress (Table 1). Water shortage significantly (P ≤ 0.05) decreased the glycine betaine (GB) contents of broccoli plants while exogenously applied NO significantly (P ≤ 0.05) enhanced the GB content of broccoli plants under water shortage (Table 1). It was noticed that foliar fed plants showed more improvement in GB contents than did the pre-sowing plants (Fig. 2). Water deficiency notably (P ≤ 0.001) increased the ascorbic acid (AsA) and total total phenolics content of broccoli plants. Exogenous application of NO was effective as pre-sowing of NO on AsA contents and both modes (pre-sowing and foliar) for total phenolics of broccoli plants under drought stress (Fig. 2). Water deficit conditions significantly increased H2O2 (P ≤ 0.01) contents of broccoli plants (Table 1). However, exogenously applied NO had no marked effect on H2O2 contents of broccoli plants under water deficit conditions (Table 1). Water deficiency and exogenous application of NO had no significant effect on total soluble proteins of broccoli plants (Table 1; Fig. 2). A significant increase was observed in the activity of SOD enzyme (P ≤ 0.001) of broccoli plants under water limited conditions (Table 1). Exogenous application of NO significantly affected the activity of SOD enzyme (P ≤ 0.05) of broccoli plants under water deficit conditions (Table 1). Foliar-applied NO had more prominent effect on broccoli plants as compared to pre-sowing seed treatment of NO under control and water deficiency (Fig. 3). Drought stress had no marked effect on the activity of POD enzyme in broccoli plants (Table 1). However, exogenously applied NO significantly affected he activity of POD (P ≤ 0.05) of broccoli plants under drought stress (Table 1). Foliar application of NO had more pronounced effects on broccoli plants as compared to the pre-sowing of NO under well-watered and water deficit conditions (Fig. 3). Water shortage and exogenous application of NO slightly increased the activity of CAT enzyme in both stressed and unstressed broccoli
peroxidase (POD) and superoxide dismutase (SOD). The activities of CAT and POD enzymes were determined using the protocol of Chance and Maehly (1955) while SOD according to Van Rossum et al. (1997). For CAT activity, the supernatant (0.1 ml) was treated with 1 ml H2O2 (5.9 mM) and 2.8 ml potassium phosphate buffer (7.8 pH; 50 mM) in quartz cuvette and read the absorbance at 240 nm after every 20 s till 180 s with the help of a spectrophotometer. For POD activity, 50 μl supernatant, 750 μl potassium phosphate buffer (pH 7.8, 50 mM) followed by100 μl guiacol (20 mM) and 100 μl H2O2 (40 mM) were added in a cuvette and read the absorbance at 470 nm after every 20 s up to 120 s on a spectrophotometer. For SOD activity, to an aliquot of 50 μl of the supernatant, 400 μl distilled water, 50 μl NBT, 50 μl riboflavin (0.026 g in 30 ml buffer having pH 7.8), 250 μl potassium phosphate buffer (pH 7.8, 50 mM), 100 μl methionine (0.44 g in 30 ml buffer having pH 7.8) and 100 μl Triton-X (75 μl in 30 ml buffer solution) were added sequentially in a plastic cuvette, placed all cuvettes containing samples under light for 15 min and read the absorbance at 560 nm using a spectrophotometer. 2.9. Statistical analysis A two-factor ANOVA was worked from the data of each attribute using the statistical software Cohort Costat program. Mean data were compared using least significance difference at 0.05% probability level. 3. Results Drought stress (60% FC) significantly reduced the shoot fresh and dry weights (P ≤ 0.01, 0.001) and shoot length (P ≤ 0.001) of broccoli (Brassica oleracea L.) plants (Table 1). However, exogenously applied nitric oxide (0.2 mM NO) considerably enhanced the shoot fresh and dry weights (P ≤ 0.01, P ≤ 0.001), and shoot length (P ≤ 0.001) of broccoli plants under drought conditions (Table 1). Foliar-applied NO showed more pronounced results as compared to pre-sowing application of NO on broccoli plants under both control and water deficit conditions (Fig. 1). Water deficit conditions markedly reduced chlorophyll a (P ≤ 0.001) and b (P ≤ 0.01) contents of broccoli plants (Table 1). While exogenous application of NO improved significantly chlorophyll a (P ≤ 0.001), and chlorophyll b (P ≤ 0.05) contents of broccoli plants under
Table 1 Mean squares from two way analysis of variance of different morphological and biochemical parameters of 72 days old broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under well watered (control, 100% FC) and water deficit (60% FC) conditions. Source of variations
df
Shoot FW
Shoot DW
Shoot length
Chlorophyll a
Drought stress (D) Nitric oxide (NO) D × NO Error
1 2 2 18
Drought (D) Nitric oxide (NO) D × NO Error
1 2 2 18
Drought (D) Nitric oxide (NO) D × NO Error
1 2 2 18
0.0415*** 0.0148*** 0.0055* 0.0013 AsA 158.4*** 19.7082ns 2.7543ns 6.8216 SOD 248.2*** 71.61* 1.588ns 15.63
1 2 2 18
0.1962*** 0.1109*** 0.0298* 0.0075 Proline 1.5084ns 4.4056ns 1.7277ns 1.8546 H2O2 515606.8** 83889.4ns 16534.1ns 44461.9 CAT 0.3609* 0.0745ns 0.0093ns 0.123
50.750*** 21.0004*** 2.3654ns 1.7331 GB 140.373* 152.395* 4.8300ns 25.5433 TSP 0.2571ns 1.2262* 0.0071ns 0.3254
Drought (D) Nitric oxide (NO) D × NO Error
2.6867** 2.1362** 0.1260ns 0.2235 Chlorophyll b 0.8050** 0.3195* 0.0461ns 0.0838 Total Phenolics 92.82ns 118.325* 1.5275ns 24.74 POD 0.0016ns 0.0067*** 0.0017* 4.9769
ns=non-significant; *, ** and *** = significant at 0.05, 0.01 and 0.001 levels, respectively. DW, Dry weight; FW, Fresh weight; GB, Glycinebetaine; AsA, Ascorbic acid; H2O2, Hydrogen peroxide; TSP, Total soluble proteins; SOD, Superoxide dismutase; POD, Peroxidase; CAT, Catalase. 9
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Fig. 1. Fresh and dry weights and lengths of shoot, chlorophyll a and b contents of broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under control and drought.
acids in plant cell metabolism (Souri and Hatamian, 2019). It is a fundamental and basic fact in plant science that chlorophyll molecules are essential for photosynthesis, but water shortage can destroy chlorophyll molecules and inhibit their synthesis, which in turn reduces photosynthesis rate in plants (Heba and Samia, 2014). In our study, water shortage decreased the chlorophyll contents of broccoli plants, analogous to what has been observed in soybean (Heba and Samia, 2014), wheat (Raza et al., 2016), radish (Akram et al., 2016) and cucumber (Naz et al., 2016) plants. They observed that drought stress decreased the chlorophyll contents of plants which thereby reduced the photosynthetic rate. However, exogenous application of NO significantly improved chlorophyll contents of broccoli plants under water shortage. Similar findings were observed in cucumber (Fan et al., 2007), rice (Farooq et al., 2009), Malus hupehensis (Cao et al., 2011), Malus rootstocks (Zhang et al., 2016). It has been reported that NO can enhance the synthesis complexes and protein molecules of chloroplasts and mitochondria (Simontacchi et al., 2015). It is now well evident that glycine betaine (GB) accumulates in plants under water shortage and is involved in improving the defensive antioxidant system by scavenging the reactive oxygen species (ROS)
plants (Table 1).
4. Discussion This study was carried out to assess whether or not and which mode of exogenously applied nitric oxide (0.02 mM) might improve oxidative defense in broccoli (Brassica oleracea L.) plants under drought stress. In the current study, water deficiency reduced the dry and fresh weight of shoots and shoot length of broccoli plants which is similar to the findings already reported in cauliflower (Latif et al., 2016; Mukhtar et al., 2016), cucumber (Naz et al., 2016) and carrot (Razzaq et al., 2017). The drought induced reduction in all these crops was ascribed to lipid peroxidation, alteration in hormone levels and nutrition uptake. However, in the current study exogenously applied nitric oxide enhanced shoot fresh and dry weights and shoot length of broccoli plants. It is similar to what has earlier been observed in wheat (Tian and Lei, 2006) and cucumber (Arasimowicz-Jelonek et al., 2009). NO is believed to enhance vegetative growth and root morphology of plants (Shao et al., 2010). Many studies show the semi hormonal effects of various nitrogenous compounds including nitrate, ammonium and some amino 10
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Fig. 2. Leaf proline, glycine betaine (GB), ascorbic acid (AsA), total phenolics, hydrogen peroxide and total soluble protein (TSP) contents of broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under control and drought stress (Mean ± S.E.) Letters (A–C) showing least significance difference among mean values.
deficit conditions was observed. Our results are also in contrast to those in quinoa (Aziz et al., 2018) wherein an increase in total phenolics under drought conditions was observed. However, exogenous application of NO enhanced the total phenolics of broccoli plants under drought stress. The increase in the total phenolics may be attributed to the fact that NO acts as a signaling molecule to activate the antioxidant defense system (Simontacchi et al., 2015). Plants possess enzymatic and non-enzymatic antioxidative defense system to protect themselves from stresses including that caused by water deficit conditions (Akram et al., 2017). In our study, drought stress increased the activity of SOD and CAT, but had no effect on POD enzyme of broccoli plants under water deficiency. Our findings for the SOD enzyme are similar but for the POD are in contrast to those in corn (Darvishan et al., 2013), wheat (Selote and Khanna-Chopra, 2010), and quinoa (Aziz et al., 2018), wherein enhanced activities of SOD and POD have been observed under water scarce regimes. On the other hand, exogenous application of NO improved the activities of SOD and POD of broccoli plants under water deficiency. This is similar to what has been found in turfgrasses (Boogar et al., 2014), hulless barley (Gan et al., 2015) and Malus rootstocks (Zhang et al., 2016). The increase in
(Ashraf et al., 2011; Vijayalakshmi et al., 2016). In the present study, water deficit conditions decreased the glycine betaine (GB) contents of broccoli plants, which is contrary to what has been observed in wheat (Singh and Bhardwaj, 2016) and quinoa (Aziz et al., 2018), wherein enhanced levels of GB have been found under water deficit conditions. Nonetheless, exogenously applied NO enhanced the GB content of broccoli plants under water shortage, analogous to what has been found in maize (Zhang et al., 2012). Zhang et al. (2012) have reported that exogenous application of NO enhanced the content and activity of choline and betaine aldehyde dehydrogenase, which are involved in GB synthesis (Sithtisarn et al., 2007). In addition, it has been shown that exogenous application of glycine amino acid alone or in form of chelated with nutrients can enhance plant growth particularly under adverse climatic conditions (Souri and Hatamian, 2019, 2019). Under oxidative stress induced by drought stress, phenolic compounds are accumulated, which can safeguard fatty acids (Amri et al., 2017). In this study, water deficit conditions did not affect the total phenolic content in broccoli plants. Our findings are in contrast to those reported in maize (Moharramnejad et al., 2015) and canola (Dawood and Sadak, 2014) wherein a decrease in total phenolics under water 11
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Fig. 3. Activities of superoxide dismutase (SOD), catalse (CAT) and peroxidase (POD) enzymes of broccoli (Brassica oleracea L.) plants treated with nitric oxide as a pre-sowing and foliar spray under control and drought stress (Mean ± S.E.), letters (A-C) showing least significance difference among mean values.
antioxidant activities could be due to the role of NO in increasing the capability of cellular antioxidative defense system (Santa-Cruz et al., 2010). Overall, water deficit conditions decreased the growth, glycine betaine, chlorophyll contents, while it enhanced ascorbic acid (AsA), hydrogen peroxide and SOD and CAT enzymes activities. Drought stress did not affect the contents of proline, total phenolics, total soluble proteins as well as the activities of POD enzyme. Exogenously applied NO enhanced fresh and dry weights of shoots, shoot length, chlorophyll contents, GB, total phenolics, total soluble proteins and the activities of CAT, SOD and POD in broccoli plants under water deficiency. However, exogenous application of NO had no effect on free proline content, AsA and H2O2 of broccoli plants under water deficit conditions. It was also observed that foliar application of NO was more effective in enhancing the drought tolerance in broccoli plants as compared to pre-sowing application of NO. Therefore, foliar application of NO could be beneficial in enhancing the oxidative defense system of broccoli plants under water deficit conditions.
249–296. Asrar, A.W.A., Elhindi, K.M., 2011. Alleviation of drought stress of marigold (Tagetes erecta) plants by using arbuscular mychorrhizal fungi. Saudi J. Biol. Sci. 18, 93–98. Aziz, A., Akram, N.A., Ashraf, M., 2018. Influence of natural and synthetic vitamin C (ascorbic acid) on primary and secondary metabolites and associated metabolism in quinoa (Chenopodium quinoa Willd.) plants under water deficit regimes. Plant Physiol. Biochem. 123, 192–203. Bates, L.S., Waldren, R.P., Teare, L.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Boogar, A.R., Salehi, H., Jowkar, A., 2014. Exogenous nitric oxide alleviates oxidative damage in turfgrasses under drought stress. S. Afr. J. Bot. 92, 78–82. Buet, A., Simontacchi, M., 2015. Nitric oxide and plant iron homeostasis. Ann. New York Acad. Sci. 1340, 39–46. Cao, H., Wang, X.W., Zou, Y.M., Shu, H.R., 2011. Effects of exogenous nitric oxide on chlorophyll fluorescence parameters and photosynthesis rate in Malus hupehensis seedlings under water stress. Acta Hort. Sin. 38, 613–620. Chance, B., Maehly, A., 1955. Assay of catalase and peroxidase. Meth. Enzymol. 2, 764–817. Chen, K., Chen, L., Fan, J., Fu, J., 2013. Alleviation of heat damage to photosystem II by nitric oxide in tall fescue. Photosyn. Res. 116, 21–31. Darvishan, M., Tohidi-Moghadam, H.R., Zahedi, H., 2013. The effect of foliar application of ascorbic acid (vitamin C) on physiological and biochemical changes of corn (Zea mays L.) under irrigation withholding in different growth stages. Maydica 58, 195–200. Dawood, M.G., Sadak, M.S., 2014. Physiological role of glycine betaine in alleviating the deleterious effects of drought stress on canola plants (Brassica napus L.). Mid. East J. Agric. Res. 3, 943–954. De Figueiredo, S.M., Binda, N.S., Nogueira-Machado, J.A., Vieira-Filho, S.A., Caligiorne, R.B., 2015. The antioxidant properties of organo-sulfur compounds (sulforaphane). Rec. Pat. Endocr. Metab Immune Drug Discov. 9, 24–33. Fan, H., Guo, S., Jiao, Y., Zhang, R., Li, J., 2007. Effects of exogenous nitric oxide on growth, active oxygen species metabolism, and photosynthetic characteristics in cucumber seedlings under NaCl stress. Front. Agric. China 1, 308–314. Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., Basra, S.M.A., 2009. Plant drought stress effects, mechanisms and management. Agron. Sustain. Develop. 29, 185–212. Fernández-Marcos, M., Sanz, L., Lewis, D.R., Muday, G.K., Lorenzo, O., 2011. Nitric oxide causes root apical meristem defects and growth inhibition while reducing PINFORMED 1 (PIN1)-dependent acropetal auxin transport. Proc. Nat. Acad. Sci. 108, 18506–18511. Galahitigama, G.A.H., Wathugala, D.L., 2016. Pre-sowing seed treatments improves the growth and drought tolerance of rice (Oryza sativa L.). Imp. J. Int. Res. 2, 1074. Gan, L., Wu, X., Zhong, Y., 2015. Exogenously applied nitric oxide enhances the drought tolerance in hulless barley. Plant Prod. Sci. 18, 52–56. Grieve, C.M., Grattan, S.R., 1983. Rapid assay for the determination of water soluble quaternary ammonium compounds. Plant Soil 70, 303–307.
References Akram, N.A., Waseem, M., Ameen, R., Ashraf, M., 2016. Trehalose pretreatment induces drought tolerance in radish (Raphanus sativus L.) plants: some key physio-biochemical traits. Acta Physiol. Plant. 38, 3. Akram, N.A., Shafiq, F., Ashraf, M., 2017. Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front. Plant Sci. 8, 613. Akram, N.A., Shafiq, F., Ashraf, M., 2018. Peanut (Arachis hypogaea L.): a prospective legume crop to offer multiple health benefits under changing climate. Compr. Rev. Food Sci. Food Saf. 17 (5), 1325–1338. Amri, Z., Lazreg-Aref, H., Mekni, M., El-Gharbi, S., Dabbaghi, O., Mechri, B., Hammami, M., 2017. Oil characterization and lipids class composition of pomegranate seeds. Biol. Med. Res. Int Article No. 8. Arasimowicz-Jelonek, M., Floryszak-Wieczorek, J., Kubiś, J., 2009. Involvement of nitric oxide in water stress-induced responses of cucumber roots. Plant Sci. 177, 682–690. Arnon, D.T., 1949. Copper enzyme in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Ashraf, M., Akram, N.A., Al-Qurainy, F., Foolad, M.R., 2011. Drought tolerance: roles of organic osmolytes, growth regulators, and mineral nutrients. Adv. Agron. 111,
12
Scientia Horticulturae 254 (2019) 7–13
A. Munawar, et al.
defense system in carrot (Daucus carota L.) plants. Sci. Hort. 225, 373–379. Santa-Cruz, D.M., Pacienza, N.A., Polizio, A.H., Balestrasse, K.B., Tomaro, M.L., Yannarelli, G.G., 2010. Nitric oxide synthase-like dependent NO production enhances heme oxygenase up-regulation in ultraviolet-B-irradiated soybean plants. Phytochemistry 71, 1700–1707. Sanz, L., Albertos, P., Mateos, I., Sánchez-Vicente, I., Lechón, T., Fernández-Marcos, M., Lorenzo, O., 2015. Nitric oxide (NO) and phytohormones crosstalk during early plant development. J. Exp. Bot. 66, 2857–2868. Selote, D.S., Khanna-Chopra, R., 2010. Antioxidant response of wheat roots to drought acclimation. Protoplasma 245, 153–163. Shao, Y., Zhang, S., Engelhard, M.H., Li, G., Shao, G., Wang, Y., Liu, J., Aksay, I.A., Lin, Y., 2010. Nitrogen-doped graphene and its electrochemical applications. J. Mater. Chem. 20, 7491–7496. Simontacchi, M., Galatro, A., Ramos-Artuso, F., Santa-María, G.E., 2015. Plant survival in a changing environment: the role of nitric oxide in plant responses to abiotic stress. Front. Plant Sci. 6, 977. Singh, N., Bhardwaj, R.D., 2016. Ascorbic acid alleviates water deficit induced growth inhibition in wheat seedlings by modulating levels of endogenous antioxidants. Biologia 71, 402–413. Sithtisarn, S., Harinasut, P., Pornbunlualap, S., Cha-um, S., 2007. Accumulation of Glycinebetaine and Betaine Aldehyde Dehydrogenase Activity in Eucalyptus camaldulensis Clone T5 Under in Vitro Salt Stress. Doctoral dissertation. Kasetsart University. Sotelo, T., Soengas, P., Velasco, P., Rodríguez, V.M., Cartea, M.E., 2014. Identification of metabolic QTLs and candidate genes for glucosinolate synthesis in Brassica oleracea leaves, seeds and flower buds. PLoS One 9, 91428. Souri, M.K., Hatamian, M., 2019. Aminochelates in plant nutrition; a review. J. Plant Nutr. 42, 67–78. Tian, X., Lei, Y., 2006. Nitric oxide treatment alleviates drought stress in wheat seedlings. Biol. Plant. 50, 775–778. Van Rossum, M.W., Alberda, M., Van der Plas, L.H., 1997. Role of oxidative damage in tulip bulb scale micropropagation. Plant Sci. 130, 207–216. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Sci. 151, 59–66. Vijayalakshmi, T., Vijayakumar, A.S., Kiranmai, K., Nareshkumar, A., Sudhakar, C., 2016. Salt stress induced modulations in growth, compatible solutes and antioxidant enzymes response in two cultivars of safflower (Carthamustinctorius L. cultivar TSF1 and cultivar SM) differing in salt tolerance. Am. J. Plant Sci. 7, 1802. Wani, S.H., Kumar, V., Shriram, V., Sah, S.K., 2016. Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J. 4, 162–176. Yuan, G.F., Sun, B., Yuan, J., Wang, Q.M., 2009. Effects of different cooking methods on health promoting compounds of broccoli. J. Zhejiang Univ. Sci. B 10, 580–588. Zhang, L.X., Zhai, Y.Y., Li, Y., Zhao, Y.O., Lv, L.X., Gao, M., Liu, J.C., Hu, J.J., 2012. Effects of nitrogen forms and drought stress on growth, photosynthesis and some physico-chemical properties of stem juice of two maize cultivars (Zea mays L.) at elongation stage. Pak. J. Bot. 44, 1405–1412. Zhang, L., Li, X., Li, X., Wei, Z., Han, M., Zhang, L., Li, B., 2016. Exogenous nitric oxide protects against drought-induced oxidative stress in Malus rootstocks. Turk. J. Bot. 40, 17–27.
Hatamian, M., Nejad, A.R., Kafi, M., Souri, M.K., Shahbazi, K., 2018. Interactions of lead and nitrate on growth characteristics of ornamental judas tree (Cercis siliquastrum). Open Agric. 3, 386–392. Heba, I.M., Samia, A.A., 2014. Influence of garlic extract on enzymatic and non-enzymatic antioxidants in soybean plants (Glycine max L.) grown under drought stress. J. Life Sci. 11, 47–58. Hichri, I., Boscari, A., Castella, C., Rovere, M., Puppo, A., Brouquisse, R., 2015. Nitric oxide: a multifaceted regulator of the nitrogen-fixing symbiosis. J. Exp. Bot. 66, 2877–2887. Jeffery, E.H., Araya, M., 2009. Physiological effects of broccoli consumption. Phytochem. Rev. 8, 283–298. Julkenen-Titto, R., 1985. Phenolic constituents in the leaves of northern willows: Methods for the analysis of certain phenolics. J. Agric. Food Chem. 33, 213–217. Khan, M.A.M., Ulrichs, C., Mewis, I., 2010. Effects of water stress and aphid herbivory flavonoids in broccoli (Brassica oleracea var. italic Plenck). J. Appl. Bot. Food Qual. 84, 178–182. Krasuska, U., Ciacka, K., Andryka-Dudek, P., Bogatek, R., Gniazdowska, A., 2015. “Nitrosative Door” in seed dormancy alleviation and germination. React. Oxygen Nitrogen Species Signal. Commun. Plants 215–237. Latif, M., Akram, N.A., Ashraf, M., 2016. Regulation of some biochemical attributes in drought-stressed cauliflower (Brassica oleracea L.) by seed pre-treatment with ascorbic acid. J. Hort. Sci. Biotechnol. 91, 129–137. Liu, F., Guo, F.Q., 2013. Nitric oxide deficiency accelerates chlorophyll breakdown and stability loss of thylakoid membranes during dark-induced leaf senescence in Arabidopsis. PLoS One 8, e56345. Lombardo, M.C., Graziano, M., Polacco, J.C., Lamattina, L., 2006. Nitric oxide functions as a positive regulator of root hair development. Plant Signal. Behav. 1, 28–33. López-Berenguer, C., García-Viguera, C., Carvajal, M., 2006. Are root hydraulic conductivity responses to salinity controlled by aquaporins in broccoli plants? Plant Soil 279, 13–23. Lozano-Juste, J., León, J., 2011. Nitric oxide regulates DELLA content and PIF expression to promote photomorphogenesis in Arabidopsis. Plant Physiol. 156, 1410–1423. Moharramnejad, S., Sofalian, O., Valizadeh, M., Asgari, A., Shiri, M., 2015. Proline, glycine betaine, total phenolics and pigment contents in response to osmotic stress in maize seedlings. J. Biosci. Biotechnol. 4. Mukherjee, S.P., Choudhuri, M.A., 1983. Implication of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol. Plant. 58, 166–170. Mukhtar, A., Akram, N.A., Aisha, R., Shafiq, S., Ashraf, M., 2016. Foliar-applied ascorbic acid enhances antioxidative potential and drought tolerance in cauliflower (Brassica oleracea L. var. Botrytis). Agrochimica 60, 107–113. Naz, H., Akram, N.A., Ashraf, M., 2016. Impact of ascorbic acid on growth and some physiological attributes of cucumber (Cucumis sativus) plants under water-deficit conditions. Pak. J. Bot. 48, 877–883. Ravikumar, C., 2015. Therapeutic potential of Brassica oleracea (broccoli)-a review. Int. J. Drug Dev. Res. 7, 9–10. Raza, M.A.S., Saleem, M.F., Khan, I.H., Shah, G.M., Raza, A., 2016. Bio-economics of foliar applied GB and K on drought stressed wheat (Triticum aestivum L.). ARPN J. Agric. Biol Sci. 11, 1. Razzaq, M., Akram, N.A., Ashraf, M., Naz, H., Al-Qurainy, F., 2017. Interactive effect of drought and nitrogen on growth, some key physiological attributes and oxidative
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