Effect of salicylic acid and sodium nitroprusside on growth parameters, photosynthetic pigments and secondary metabolites of safflower under drought stress

Scientia Horticulturae 259 (2020) 108823

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Effect of salicylic acid and sodium nitroprusside on growth parameters, photosynthetic pigments and secondary metabolites of safflower under drought stress

T

⁎

Maryam Chavoushia, , Farzaneh Najafia, Azam Salimia, Seyed Abdolhamid Angajib a b

Department of Plant Science, Faculty of Biological Science, Kharazmi University, P.O. Box: 15719-14911, Tehran, Iran Department of Cell and Molecular Biology, Faculty of Biological Science, Kharazmi University, Tehran, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Drought stress Nitric oxide Photosynthetic pigments Secondary metabolites Salicylic acid

The effect of drought stress on the production of secondary metabolites and photosynthetic pigments in safflower, Carthamus tinctorius L., was studied in the present work. The application of two regulating signaling molecules of salicylic acid (SA) and sodium nitroprusside (SNP) were respectively examined at concentrations of 250 μM and 25 μM on the plants subjected to different field capacity (FC) volumes of water (25% and 100%). The drought stress decreased the leaf number and area, shoot fresh and dry weights, and shoot length, whereas the root length and fresh weight were increased. There was an increase in the secondary metabolites (flavonoids, anthocyanin, phenol, and phenylalanine ammonia lyase activity) and a decrease in photosynthetic pigments. The SA treatment improved the rate of photosynthesis, anthocyanin content, and phenylalanine ammonia lyase activity. The SNP treatment improved the growth of aerial parts and hindered the root elongation possibly because of their free radical scavenging ability. The drought stress can be applied for enhanced production of secondary metabolites and the simultaneous administration of SA and SNP can be proposed as a mitigation strategy to reserve the normal plant function.

1. Introduction Safflower (Carthamus tinctorius L.), a member of Asteraceae family, is an oil seed crop and a source of coloring agent in food industry (Ekin, 2005). Safflower flowers are also well known for their medicinal properties (Ekin, 2005). In recent years, the climatic changes and water scarcity problems negatively affected the productivity of these plant species in semi-arid regions. In addition, the need to efficient use of water resources became highly important because of the anticipated water scarcity in the face of global warming and the increasing competition between domestic, industrial, and agricultural water consumptions (Zali and Ehsanzadeh, 2018). The drought is one of the main factors limiting the growth, cell expansion, stomatal conductance, biomass, and crop production (Gunes et al., 2007). To improve plant tolerance to drought stress, the exogenous addition of signaling molecules such as salicylic acid (SA) and sodium nitroprusside (SNP) was introduced as a commonly applied and well-known mitigation strategy (Ghadakchiasl and Mozafari, 2017; Yadu et al., 2017). The nitrogen monoxide (NO) is a bioactive molecule involved in the regulation of diverse physiological processes such as germination,

⁎

metabolism, cell death, opening and closing of stomata, and root growth (Misra et al., 2010). The NO can be generated through the chemical breakdown of NO donor molecules, such as SNP, with the chemical formula of Na2[Fe(CN)5NO] (Dieckmann et al., 2012; Neill et al., 2003). The SNP releases NO molecule upon light irradiation (Floryszak-Wieczorek et al., 2006). The NO is a small gaseous molecule with hydrophobic properties. It can easily diffuse thorough cell membranes and reach intracellular targets (Arasimowicz and FloryszakWieczorek, 2007). Thus, the NO not only can easily migrate to the hydrophilic regions of the cell, such as the cytoplasm, but also can freely diffuse through the lipid phase of membranes and act as a cellular signaling system between cells (Arasimowicz and Floryszak-Wieczorek, 2007). The low NO concentrations in plants might rapidly eliminate lipid peroxyl radicals and alter the species and components of reactive oxygen species (ROS). Accordingly, it blocks the injury from ROS and affects the expression of antioxidant genes (Lamotte et al., 2006). There are reports on the NO stimulation of the defensive mechanisms in wheat and reduction of the oxidative damage by improving the antioxidant system and maintaining ROS balance of the plants under osmotic stress (Khan et al., 2017)

Corresponding author. E-mail addresses: [email protected], [email protected] (M. Chavoushi).

https://doi.org/10.1016/j.scienta.2019.108823 Received 6 July 2019; Received in revised form 21 August 2019; Accepted 31 August 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Scientia Horticulturae 259 (2020) 108823

M. Chavoushi, et al.

Salicylic acid is also known as endogenous growth regulator of physiological processes, such as growth and development, photosynthesis, and root development and germination (Miura and Tada, 2014). It stimulates the root development and stomata closure as well as induces the expression of defense genes and secondary metabolites (Miura and Tada, 2014). The effect of SA depends on the plant species, functional model (including spraying used in hydroponics), concentration (El-Tayeb, 2005), and the length of treatment (Qados, 2015). The NO and SA are able to protect plants from diverse biotic and abiotic stresses such as high temperatures, drought, salt stress, and pathogen (Miura and Tada, 2014; Siddiqui et al., 2011). Salicylic acid treatment of salt-stressed cultivators of mungbean increased nitrogen and sulfur assimilation, glutathione content, and the activity of ascorbate peroxidase and glutathione reductase. It also caused the accumulation of glycine betaine that in combination with reduced ethylene increased the glutathione content and reduced the oxidative stress. In overall, the enhanced photosynthesis and growth of the plant did occur in salt stressed cells treated with both 10 and 1000 μM SA (Khan et al., 2014). The plants accumulate several secondary metabolites in response to drought stress. The increase in osmolytes such as soluble sugars and proline is one of the other well-known adaptive solutions by the plant species. Many studies on the role of osmoregulating chemicals and phytohormone under drought stress conditions have been done (Ghobadi et al., 2013; Xiao et al., 2008), but the metabolic profiles of the photosynthetic pigments and secondary metabolites still need further studies. The objective of the present research is to comprehensively examine the effects of drought stress as well as the NO and SA treatment on the photosynthetic pigments and secondary metabolites of safflower.

2.3. Determination of secondary metabolites 2.3.1. Total phenol The total phenol content was determined by the method of (Singleton and Rossi, 1965). According to this method, the leaf samples were grounded to homogenization at 4 °C with 1 mL of methanol (80%) and then centrifuged at 15,000 × gfor 15 min. The extract was mixed with 0.5 mL of the Folin-Ciocalteu reagent (diluted 1:10 with water), followed by addition of 1 mL of a 5% sodium carbonate solution. After 30 min, the absorbance was read at 725 nm. Total phenol was determined by a calibration curve method and expressed as mg Gallic acid equivalents. 2.4. Anthocyanin The leaf samples were grounded with acidic methanol at 4 °C and centrifuged at 15,000 × gfor 15 min. The extract was added to 100 μL of chloroform. The absorbance was then measured at 530 nm and expressed as mg g−1 FW (Diaz et al., 2006). 2.5. Flavonoid The total flavonoid content was determined by the aluminum chloride colorimetric assay. The leaf samples were grounded with methanol and then 0.30 mL of 5% NaNO2 was added to 1 mL aliquots of the extract. After 5 min, 0.3 mL of 10% AlCl3 was added. After further 5 min, 2 mL of 1 M NaOH was added and the volume was filled up to 10 mL with distilled water. The solution was mixed and its absorbance was measured against a blank at 510 nm (Toor and Savage, 2005). The flavonoid content was quantified by a calibration curve method using quercetin as the standard of flavonoid compounds.

2. Material and methods 2.1. Culture condition and treatments

2.6. Phenylalanine ammonia lyase (PAL) activity

Safflower seeds were obtained from the Oil Seed Section, Seed and Plant Improvement Institute, Karaj (Alborz, Iran). The seeds were sterilized with sodium hypochlorite (1%) for 5 min and then washed several times by distilled water. The uniform seeds germinated after 72 h at 25 °C and the seedlings were transferred to pots containing sand, clay, and humus (3:1:2) under a light density of approximately − 100 μmol m−2 s 1, day/night temperatures of 26/18 °C under 16 h photoperiod. The 23 days old plants were treated with SA (0 and 250 μM) and SNP (0 and 25 μM). After 24 h, the plants were exposed to the 25% (drought condition) and 100% field capacity (FC) volumes of water. After seven days, the plants were again sprayed with the SNP and SA. The plants were then harvested after 14 days and the length of shoots (SL), roots (RL), leaf area (LA), and leaf number (LN) were measured and the fresh weights (FWs) of total plants (shoots and roots) were recorded. To determine dry weights (DWs), the fresh plants were dried in oven at 70 °C and weighed after reaching a constant mass (72 h).

The samples of leaves were grounded to homogenization by 200 mM sodium phosphate buffer pH 6.2 containing 5% (w/v) polyvinyl pyrrolidone and 2% Triton X-100. The mixture was centrifuged at 15,000 × gfor 20 min at 4 °C. To measure the activity of the enzyme mixtures containing 50 mM Tris buffer (pH 8.8), a 600 μL phenylalanine (2 mM) and 800 μL of the extract were prepared. The resulting mixture was left at 37 °C for 1 h. The reaction was stopped by adding 100 μL of 2 M HCl. In the next step, 2 mL of toluene was added and the reaction mixture was mixed by a vortex device for 30 s. The mixture was then centrifuged at 5000 rpm for 5 min. The supernatant absorbance was measured at 290 nm (Wang et al., 2006). 2.7. Flavonoid fractionation A dried leaf (0.5 g) was homogenized with 40 mL of 62.5% aqueous methanol containing 2 g L−1 of 2, (3)-tert-butyl-4-hydroxyanisole (BHA). The mixture was then ultrasonicated for 5 min. To this extract, 10 mL of 6 M HCl was added and put it in water bath at 90 °C for 2 h. The sample was allowed to cool and then it was filtered. The separation was done on a 25 cm × 4.6 mm with a pre-column, Eurospher 100-5 C18 analytical column provided by Knauer (Berlin, Germany). The data acquisition and integration was performed with EZchrom Elite. The peaks were monitored at 260–250–268–370 nm wavelength. The injection volume was 20 μL and the temperature was maintained at 25 °C (Mattila et al., 2000).

2.2. The content of photosynthetic pigments The content of photosynthetic pigments including chlorophyll a, b (Chl a, Chl b) and carotenoids were determined according to the method of Lichtenthaler (1987). The fresh leaves (0.5 g) were homogenized in acetone (80%) and then centrifuged at 15,000 × gfor 30 min. The absorbance of supernatant was recorded at the wavelengths of 646.8, 663.2, and 470 nm. The contents of chlorophyll and carotenoids were then calculated by the following empirical correlations (Lichtenthaler, 1987). Chl a (g L−1) = 12.25 A663.2 – 2.79 A646.8

(1)

Chl b (g L−1) = 21.51 A646.8 – 5.10 A663.2

(2)

Cx+c (g L−1) = (1000 A470 – 1.8 Chl a – 85.52 Chl b) /198

(3)

2.8. Statistical analysis All the experiments were done in triplicates unless otherwise specified. The experiments were arranged in a randomized complete block design with three replications for each test. For comparing significant 2

Scientia Horticulturae 259 (2020) 108823

M. Chavoushi, et al.

Table 1 Effect of SA, SNP, and drought stress on the shoot length (SL), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), root dry weight (RDW), leaf number (LN), and leaf area (LA) of Carthamus tinctorius L. Values with similar letters are not significantly different at p < 0.05. Treatments

SL (cm)

FC FC FC FC FC FC FC FC

14.0 8.00 12.3 11.0 11.6 11.5 12.0 10.0

100% 25% 100% + SA 25% + SA 100% + SNP 25% + SNP 100% + SA + SNP 25% + SA + SNP

a d ab bc bc bc b c

RL (cm)

SFW (g)

9.0 cd 13.3 ab 9.6 cd 14.6 a 11.3 bc 11.0 c 8.3 d 8.5 d

3.6 0.7 2.1 3.3 1.6 2.8 1.6 3.3

SDW (g)

a

0.28 0.05 0.32 0.27 0.13 0.18 0.14 0.26

d c ab c b c ab

a c a a ab ab ab a

RFW (g) 0.83 1.20 0.48 1.20 0.96 0.64 0.93 0.65

bc a d a b cd b cd

RDW (g) 0.06 0.04 0.03 0.08 0.05 0.04 0.07 0.04

bcd cd d a abc cd a cd

LN

LA (cm2)

7.3 cd 5.0 e 7.6bcd 7.3 cd 9.0 a 8.0 bc 8.3 ab 7.0 d

11.2 a 2.5 f 5.3 ef 7.9 bc 4.2 e 8.9 b 6.6 cd 8.6 b

(∼17%) and leaf number (∼14%), reduced shoot length (14%), shoot fresh weight (∼56%) and leaf area (∼41%) and had no significant effect on root fresh weight, root length, and shoot dry weight compared to the control, FC 100%. The supplementation of the SA and SNP to the growth media of the drought stressed plants increased the shoot length (from 8.0 cm to 10.0 cm), fresh and dry weights of the shoot (about five fold), leaf number (from 5.0 to 7.0), and leaf area (2.5 cm2 to 8.6 cm2), but decreased the root length (from 13.3 cm to 8.5 cm) and the root fresh weight (to about half) compared with the plants under drought stress (Table 1).

differences of the set of means, the Duncan test was applied at p < 0.05 using SPSS software version No. 16. (SPSS Inc. Chicago IL).

3. Results 3.1. Growth parameters The drought stress caused a reduction in the length of shoot, the number of leaves, and the leaf area. There was about five fold decrease in the fresh and dry weights of the shoot respectively from 3.6 g and 0. 28 g to 0.7 g and 0.05 g, but the length and fresh weights of the root increased about 45%. However, the drought stress had no significant impact on the dry weight of the root, Table 1. The exposure of the nonstressed plants (FC 100%) to 250 μM SA caused about 41% decrease in the root and shoot fresh weights and about 52% reduction in the leaf area. It had no significant effect on the root length and dry weight, the shoot dry weight, and the leaf number compared to their respective control. The SA treatment of the drought stressed plants did not significantly change the root fresh weight (∼1.2 g) and the root length (∼14 cm) of the plants (FC 25% + SA) compared to the drought stressed ones (FC 25%). The dry weight of the roots, shoot length, leaf area, and leaf number of the SA treated plants under drought stress were significantly increased compared to their respective controls, FC 25% (Table 2). The SNP treatment of plants at normal growth conditions (FC 100%) reduced about 17% the length and about 56% the fresh weight of the shoots. The leaf area reduced from 11.2 cm2 to 4.2 cm2 upon the SNP treatment of the non-stressed plants (Table 1), whereas the leaf number increased ∼23% from 7.3 to 9.0. This treatment had no significant effect on the root fresh weight, root length, root dry weight, and shoot dry weight in comparison with those of control (FC 100%). The SNP addition to the drought stressed plants reduced the length (17%) and fresh weight (47%) of the root compared to the drought stressed plants, FC 25% (Table 1), whereas the shoot length was increased from 8.0 cm to 11.5 cm. It had no significant effect on the root dry weight (Table 1). At non-stress conditions (FC 100%), the simultaneous application of 250 μM SA and 25 μM SNP to the plants increased the root dry weight

3.2. The content of photosynthetic pigments The contents of chlorophyll a, b and carotenoids were significantly decreased by the drought stress. The SA treatment of the non-stressed plants (FC 100%) reduced about 45% the contents of Chl a and carotenoids and had no significant effect on the Chl b compared to those of control. The SA treatment of the drought stressed plants increased Chl a, but not significantly changed the Chl b and carotenoids compared to the drought stressed plants (FC 25%). At normal growth conditions (FC 100%), the SNP treatment reduced the Chl a from 0.87 to 0.56 mg g−1 FW, but not affected the Chl b and carotenoids in comparison with control. The SNP application to the drought stressed plants increased the Chl b about 3.5 times (from 0.13 to 0.45 mg g−1 FW) and had no significant impact on Chl a and carotenoids compared with the drought stressed ones (FC 25%). The simultaneous addition of both SNP and SA to the plants at normal growth conditions (FC 100%) caused no significant changes on the contents of the photosynthetic pigments; while this treatment to the drought stressed plants increased 2.5 times the contents of Chl a, b and 3.5 times the carotenoids compared to those of the drought stressed plants (Table 2). 3.3. The contents of secondary metabolites and the activity of phenylalanine ammonia lyase (PAL) The contents of anthocyanin, flavonoids (rutin, quercetin, luteloin, and apigenin), and phenols as well as the PAL activity were

Table 2 Effect of SA, SNP, and drought stress on the activity of phenylalanine ammonia lyase (PAL) and the contents of photosynthetic pigments, anthocyanin, flavonoids, and phenol in Carthamus tinctorius L. Values with similar letters are not significantly different at p < 0.05. Treatments

Chl a (mg g−1 FW)

FC FC FC FC FC FC FC FC

0.87 0.28 0.47 0.49 0.56 0.17 0.65 0.70

100% 25% 100% + SA 25% + SA 100% + SNP 25% + SNP 100% + SA + SNP 25% + SA + SNP

a d bc bc bc d ab ab

Chl b (mg g−1 FW) 0.40 0.13 0.26 0.17 0.30 0.45 0.31 0.32

ab d bcd d bc a abc abc

Carotenoids (mg g−1 FW) 0.38 0.09 0.21 0.19 0.25 0.19 0.29 0.32

a c bc bc ab bc ab ab

mg g−1 F.W: mg per g fresh weight; U mg−1 protein: unit of activity per mg protein. 3

Anthocyanin (mg −1 FW)

Phenol (mg g−1 FW)

PAL activity (U mg−1protein)

81 cd 190 a 53 d 86 cd 103 c 114 c 160 b 157 b

108 b 130 a 86 cd 146 a 79 cd 89 bc 67 de 58 f

2.2 3.7 2.9 2.8 2.4 2.1 2.2 2.9

d a b b c d d b

Flavonoids (mg g−1 FW) 28 64 43 63 51 42 74 56

d ab c ab bc cd a bc

Scientia Horticulturae 259 (2020) 108823

M. Chavoushi, et al.

significantly increased about 1.2–2.3 times under drought stress conditions compared with normal growth conditions. The highest increase was that of anthocyanin and the least for phenols. At normal growth conditions (FC 100%), the SA treatment had no significant effect on the anthocyanin, but enhanced the PAL activity (∼32%) and the contents of flavonoids (∼54%). The SA treatment of the plants at normal growth conditions reduced ∼20% the phenol contents from 108 to 86 mg g−1 FW (Table 2). The contents of anthocyanin, rutin, quercetin, luteloin, and apigenin as well as the activity of PAL were reduced in the droughtstressed plants upon application of the SA (Figs. 1–4 and Table 2). The SNP treatment of the non-stressed plants (FC 100%) had no significant effect on the anthocyanin contents, whereas the PAL activity and the contents of flavonoids (rutin, quercetin, luteloin, and apigenin) were respectively increased about 9% and 82% (Table 2 and Figs. 1–4). This treatment reduced about 27% the phenol contents from 108 to 79 mg g−1 FW. The SNP treatment of drought stressed plants reduced the content of anthocyanin and the PAL activity about 1.7 times and the content of flavonoids and phenols about 1.5 times compared with the drought stressed ones (FC 25%). The combined application of both SNP and SA to the plants at normal growth conditions did not significantly change the PAL activity but increased anthocyanin and flavonoids (rutin, quercetin, luteloin and apigenin) respectively about 2.0 and 2.6 fold (Figs. 1–4). This treatment reduced ∼38% the phenol contents from 108 to 67 mg g−1 FW. The simultaneous application of both SA and SNP to the droughtstressed plants (FC 25%) reduced the PAL activity and the contents of anthocyanin, phenols, quercetin, and luteloin (Table 2 and Figs. 1–4). This treatment resulted in significance increase in the rutin and apigenin contents compared to the drought stressed ones (Figs. 1 and 4).

Fig. 1. Effect of SA, SNP, and drought stress on rutin content of Carthamus tinctorius L. Values with similar letters are not significantly different at p < 0.05.

4. Discussion Fig. 2. Effect of SA, SNP, and drought stress on quercetin content of Carthamus tinctorius L. Values with similar letters are not significantly different at p < 0.05.

Drought stress is an abiotic stress for the plant species so that it negatively affected the safflower growth (Table 1). The drought stress closes the stomata, reduces CO2 reduction, inhibits Calvin cycle, prevents electron transfer, and creates free radicals in the chloroplast, accompanied by reduction in plant growth and crop production yield (Parida and Das, 2005). The growth of leaf surface area and the production of dry matter by photosynthesis are important cellular functions during the vegetative plant growth. Drought stress decreases cell division and prevents cell expansion by affecting photosynthesis, respiration, absorption and transfer of materials as well as by reducing turgor pressure (Jaleel et al., 2009). The decrease in the photosynthetic pigments (Chl a, b) to about one third is consistent with this observation, Table 2. Consistently, the reduced leaf growth due to drought stress followed by reduced shoot elongation was reported in the review article (Jaleel et al., 2009). However, the drought stress positively affected the root growth so that the length and fresh weight of the root increased about 45%, Table 1. This observation could be attributed to the water shortage that is led to the extension and growth of the root for efficient uptake of soil moisture (Akinci and Losel, 2009). In the present study, the SA treatment increased the root dry weight and plantlet growth under drought stress (Table 1), which is consistent with the reported effects of salicylic acid on Phaseolus vulgaris (Sadeghipour and Aghaei, 2012). Salicylic acid treatment mitigated the plant growth under drought stress by increasing the intensity of photosynthesis and cell division (Pradhan et al., 2015). It controls root growth by affecting certain growth regulators such as ABA, ethylene (Zhu, 2001), and auxin (Pacheco et al., 2013). The SNP treatment increased the shoot length under drought in the present study. A similar result was also reported for peas and wheat (Popova and Tuan, 2010). This observation can be attributed to the effect of NO on cell growth such that the NO increases exo and endo-βD-glucanase activities of the cell wall, which breaks glycosides linkage between glucose units of the wall and increases its extensibility (Hayat et al., 2014). The SNP treatment reduced root fresh weight and root

Fig. 3. Effect of SA, SNP, and drought stress on luteloin of Carthamus tinctorius L. Values with similar letters are not significantly different at p < 0.05.

Fig. 4. Effect of SA, SNP and drought stress on apigenin content of Carthamus tinctorius L. Values with similar letters are not significantly different at p < 0.05. 4

Scientia Horticulturae 259 (2020) 108823

M. Chavoushi, et al.

Pinus and olive (Elhami et al., 2015; Sayyari et al., 2013; Zamani et al., 2014). The drought stress accompanied by osmotic shock to plants that is led to increased secondary metabolites in the species. This enhancement can be attributed to the increase in the PAL activity (Table 2), as similarly reported in literature (Esra et al., 2010). The PAL is a key enzyme in the biosynthesis of phenolics such as flavonoids and anthocyanin that lead to increased phenolics and flavonoids in response to some biotic and abiotic stresses (Şen, 2012). The phenolic compounds have a special chemical structure in plants protecting cells against oxidative stress by chelating metals and binding with free radicals accompanied by less lipid peroxidation (Michalak, 2006). Flavonoids also inhibit lipid peroxidation and increase the stability of the membrane and the membrane fluidity, preventing the release of ROS and the inhibition of the peroxidation reactions (Kadioglu et al., 2011). The SA treatment of drought stressed plants reduced their anthocyanin content and the PAL activity. This observation proposes the relationship between the PAL activity and anthocyanin biosynthesis. The SNP reduced the content of secondary metabolites and the PAL activity in drought stress as well. The SNP increased poly phenol oxidase and reduced PAL activity. Similarly, the SNP has been reported to reduce phenolic contents under salt stress in Avicennia (Chen et al., 2014). Soluble carbohydrates are necessary to phenol biosynthesis. The reduction of phenolic compounds under SNP treatment might be because of the reduced sugar content under stress conditions (Gebaly, 2007). In the present study, the secondary metabolites such as anthocyanin and phenol lowered in content upon combined application of SA and SNP to drought stressed plants. It showed that the NO scavenged ROS and consequently reduced free radicals in drought stressed plants (Lei et al., 2007). The SA and SNP treatment of the soybean plants under salinity stress increased the flavonoid and anthocyanin contents due to an increase in its free radicals (Simaei et al., 2012). However, the contents of anthocyanin, phenol, quercetin, and luteloin were decreased upon the SA and SNP treatment of safflower in the present work possibly because of the activation of other defensive pathways. Consequently, the ROS were efficiently eliminated by non-enzymatic and enzymatic antioxidants under drought stress conditions. Besides, the SNP can protect the membrane by scavenging free radicals and inhibiting the membrane's peroxidation enzymes. According to Jin et al. (2010), the SNP plays a protective role by reacting with lipid radical and inhibiting it in the development or propagation phase and thus preventing lipid peroxidation. Upon drought stress, the safflower increased enzyme antioxidant such as catalase (results not shown) and non-enzyme antioxidants such as rutin and apigenin (Figs. 1 and 4). Both of them could reduce the cell free radicals and make the plant resistant against drought stress.

length in drought stress (Table 1) due to the release of NO as a growth regulator involved in many cellular processes, affecting cell cycle and root development genes. Interestingly, the NO sounds to accumulate in cortex/endodermis stem cells in Arabidopsis root tips and inhibit PIN1dependent acropetal polar auxin transport (Domingos et al., 2015). The interaction of SA and SNP under drought stress increased the shoot growth by acidifying the wall and also through enhanced metabolite synthesis and carbon fixation (Sadeghipour and Aghaei, 2012). The photosynthetic pigments were reduced by the drought stress, Table 2. A similar observation has also been reported in Catharanthus roseus (Ahmadizadeh, 2013). Both Chl a and Chl b are sensitive to a decrease in the water potential of the soil. Chlorophyll is one of the most important parts of the photosynthetic apparatus. The reduction in chlorophyll contents under drought stress showed its possible breakdown (Xiao et al., 2008). Drought stress increased the chlorophyllase enzyme activity, changed chlorophyll structure and lipid–protein complex in the membrane and reduced the light-harvesting pigment protein associated with photosystem II and the inactive enzyme of the Calvin cycle (Ahmadizadeh, 2013). Carotenoids play a fundamental role in the photosynthesis and resistance to drought stress (Jaleel et al., 2009). They are essential molecules involved in light protection during photosynthesis. They are antioxidants and single oxygen scavengers and prevent lipid peroxidation and stabilize membranes (Xiao et al., 2008). The SA treatment of drought stressed safflower caused an increase in its Chl a contents compared to that of the drought stressed plants, (Table 2). A similar observation was reported for soybean, corn, and marigold (Khan et al., 2003). An increase in growth and rate of photosynthesis in plants sprayed with SA can be dependent on the changes of photosystem II and Rubisco enzyme activity (Pacheco et al., 2013). Interestingly, the SNP treatment of drought stressed safflower caused an increase in its Chl b contents and had no significant effect on the Chl a contents compared to that of drought stress plants, Table 2. The photosynthetic pigments are directly connected to the growth. The NO protects photosynthetic pigments and light harvesting complexes in drought condition, as reported in cotton (Shallan et al., 2012). On the other hand, the SNP preserve the relative water content, reduces free radicals such as H2O2 (Tian and Lei, 2007) and increases the activity of antioxidant enzymes (Sheokand et al., 2010). The SNP influences the Fe availability and thus enhanced the Chl b synthesis (Neill et al., 2003) and protected the D1 protein of PSII (Lei et al., 2007). The SNP increased the photosynthetic pigments in tomato under Salinity stress (Nasibi, 2011) which is in agreement with our results. In this study, the combined application of SA and SNP increased the photosynthetic pigments, as similarly reported in Arachis hypogaea. The increase in the photosynthetic pigment levels upon SA and SNP treatment can also be attributed to the facilitation in iron absorption by activating the ATPase pump (Kong et al., 2014). Accordingly, either SA or NO protected photosynthetic pigments and increased growth (Dong et al., 2015). The improvement in such growth parameters as SDW, SFW, SL, LA, and LN upon combined application of SA and SNP to drought stressed plants can also be related to increase in the photosynthetic pigments via protecting their relative activities. The SA increases the chlorophyll biosynthesis and photosynthesis and the accumulation of alpha amino levulinic acid (α-ALA) as a chlorophyll biosynthetic intermediates (Kumar et al., 2010). The interaction of SA with SNP could enhance the content of amino acids or osmolytes and scavenge hydroxyl radicals that result in the stabilization of the structure and function of macromolecules such as DNA, protein, and membranes. It has been reported that the application of SA (50 μM) and NO (100 and 200 μM) caused ABA and IAA accumulation in wheat seedlings that accompanied by the development of anti-stress reactions such as high osmolytes contents (Sakhabutdinova et al., 2003; Tan et al., 2008). In the present work, the relative water content (RWC) of plants treated either with SA, SNP, or both of them increased in drought stressed plants (results were not shown) which is in agreement with the similar observation reported in

5. Conclusion Drought stress negatively affected the growth of the safflower aerial parts. However, both fresh and dry weights of the root increased in order to circumvent the water shortage by the root extension leading to efficient uptake of soil moisture. The SA treatment of the plants mitigated drought stress through improvement of photosynthesis and activating enzymatic and non-enzymatic antioxidants (carotenoids, anthocyanin, and PAL activity). The SNP treatment improved the growth of aerial parts by protecting the photosynthetic pigments by eliminating free radicals. It decreased the root growth by regulating the cell cycle and root development genes. The drought stress caused an increase in the secondary metabolites (flavonoids, anthocyanin, phenol, and PAL activity) and a decrease in the photosynthetic pigments (carotenoids and Chl a, b). The simultaneous application of SA and SNP could better reduce the damage caused by drought stress because of enhanced production and protection of photosynthetic pigments, elimination of free radicals, and activation of antioxidant systems. 5

Scientia Horticulturae 259 (2020) 108823

M. Chavoushi, et al.

References

Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Method Enzymol. 350–382. Mattila, P., Astola, J., Kumpulainen, J., 2000. Determination of flavonoids in plant material by HPLC with diode-array and electro-array detections. Agric. Food Chem. 48, 5834–5841. Michalak, A., 2006. Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Pol. J. Environ. Stud 15, 523–530. Misra, A., Misra, M., Singh, R., 2010. Nitric oxide biochemistry, mode of action and signaling in plants. Med. Plants Res. 4, 2729–2739. Miura, K., Tada, Y., 2014. Regulation of water, salinity, and cold stress responses by salicylic acid. Front. Plant Sci. 5, 4. Nasibi, F., 2011. Effect of diffrent concentrations of sodium nitroprusside (SNP) pretratment on oxidative damages induce by drought stress in tomato plant. Plant Biol. 9, 63–74. Neill, S.J., Desikan, R., Hancock, J.T., 2003. Nitric oxide signalling in plants. New Phytol. 159, 11–35. Pacheco, A.C., da Silva Cabral, C., da Silva Fermino, E.S., Aleman, C.C., 2013. Salicylic acid induced changes to growth, flowering and flavonoids production in marigold plants. Med. Plants Res. 7, 3158–3163. Parida, A.K., Das, A.B., 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicol. Environ. Saf. 60, 324–349. Popova, L., Tuan, T., 2010. Nitric oxide in plants: properties, biosynthesis and physiological functions. Iran. J. Sci. Technol. (Sciences) 34, 173–183. Pradhan, M., Tripathy, P., Mandal, P., Sahoo, B., Pradhan, R., Mishra, S., Mishra, H., 2015. Effect of salicylic acid on growth and bulb yield of onion (Allium cepa L.). Int. J. Bioresour. Stress Manag. 960–963. Qados, A.M.A., 2015. Effects of salicylic acid on growth, yield and chemical contents of pepper (Capsicum annuum L.) plants grown under salt stress conditions. Int. J. Agric. Crop Sci. 8, 107–113. Sadeghipour, O., Aghaei, P., 2012. Impact of exogenous salicylic acid application on some traits of common bean (Phaseolus vulgaris L.) under water stress conditions. Int. J. Agric. Crop Sci. 4, 685–690. Sakhabutdinova, A., Fatkhutdinova, D., Bezrukova, M., Shakirova, F., 2003. Salicylic acid prevents the damaging action of stress factors on wheat plants. Bulg. J. Plant Physiol. 21, 314–319. Sayyari, M., Ghavami, M., Ghanbari, F., Kordi, S., 2013. Assessment of salicylic acid impacts on growth rate and some physiological parameters of lettuce plants under drought stress conditions. Int. J. Agric. Crop Sci. 5, 1951–1957. Şen, A., 2012. Oxidative stress studies in plant tissue culture, antioxidant enzyme. Techopen. pp. 59–88. Shallan, M., Hassan, H., Namich, A., Ibrahim, A., 2012. Effect of sodium nitroprusside, putrescine and glycine betaine on alleviation of drought stress in cotton plant. Am. Eurasian J. Agric. Environ. 12, 1252–1265. Sheokand, S., Bhankar, V., Sawhney, V., 2010. Ameliorative effect of exogenous nitric oxide on oxidative metabolism in NaCl treated chickpea plants. Braz. J. Plant Physiol. 22, 81–90. Siddiqui, M.H., Al-Whaibi, M.H., Basalah, M.O., 2011. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248, 447–455. Simaei, M., Khavari-Nejad, R., Bernard, F., 2012. Exogenous application of salicylic acid and nitric oxide on the ionic contents and enzymatic activities in NaCl stressed soybean plants. Am. J. Plant Sci. 3, 1495–1503. Singleton, V., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Viticult. 16, 144–158. Tan, J., Zhao, H., Hong, J., Han, Y., Li, H., Zhao, W., 2008. Effects of exogenous nitric oxide on photosynthesis, antioxidant capacity and proline accumulation in wheat seedlings subjected to osmotic stress. World J. Agric. Sci. 4, 307–313. Tian, X., Lei, Y.B., 2007. Physiological responses of wheat seedlings to drought and UV-B radiation. Effect of exogenous sodium nitroprusside application. Russ. J. Plant Physiol. 54, 676–682. Toor, R.K., Savage, G.P., 2005. Antioxidant activity in different fractions of tomatoes. Food Res. Int. 38, 487–494. Wang, J.W., Zheng, L.P., Wu, J.Y., Tan, R.X., 2006. Involvement of nitric oxide in oxidative burst, phenylalanine ammonia-lyase activation and Taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures. Nitric Oxide 15, 351–358. Xiao, X., Xu, X., Yang, F., 2008. Adaptive responses to progressive drought stress in two Populus cathayana populations. Silva Fenn. 42, 705–719. Yadu, S., Dewangan, T.L., Chandrakar, V., Keshavkant, S., 2017. Imperative roles of salicylic acid and nitric oxide in improving salinity tolerance in Pisum sativum L. Physiol. Mol. Biol. Plants 23, 43–58. Zali, A.G., Ehsanzadeh, P., 2018. Exogenous proline improves osmoregulation, physiological functions, essential oil, and seed yield of fennel. Ind. Crop Prod. 111, 133–140. Zamani, M., Hakimi, M., Mosleh Arany, A., Kiani, B., Rashtian, A., 2014. The effects of salicylic acid (SA) and sodium nitroprusside (SNP) on physical and growth characteristics of Pinus eldarica. Bull. Environ. Pharmacol. Life Sci. 3, 31–35. Zhu, J.-K., 2001. Cell signaling under salt, water and cold stresses. Curr. Opin. Plant Biol. 4, 401–406.

Ahmadizadeh, M., 2013. Physiological and agro-morphological response to drought stress. Middle-East J. Sci. Res. 13, 998–1009. Akinci, S., Losel, D.M., 2009. The soluble sugars determination in cucurbitaceae species under water stress and recovery periods. Adv. Environ. Biol. 3, 175–183. Arasimowicz, M., Floryszak-Wieczorek, J., 2007. Nitric oxide as a bioactive signalling molecule in plant stress responses. Plant Sci. 172, 876–887. Chen, J., Xiao, Q., Wang, C., Wang, W.-H., Wu, F.-H., He, B.-Y., Zhu, Z., Ru, Q.-M., Zhang, L.-L., Zheng, H.-L., 2014. Nitric oxide alleviates oxidative stress caused by salt in leaves of a mangrove species, Aegiceras corniculatum. Aquat. Bot. 117, 41–47. Diaz, C., Saliba-Colombani, V., Loudet, O., Belluomo, P., Moreau, L., Daniel-Vedele, F., Morot-Gaudry, J.-F., Masclaux-Daubresse, C., 2006. Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana. Plant Cell Physiol. 47, 74–83. Dieckmann, V., Eicke, S., Springfeld, K., Imlau, M., 2012. Transition metal compounds towards holography. Materials 5, 1155–1175. Domingos, P., Prado, A.M., Wong, A., Gehring, C., Feijo, J.A., 2015. Nitric oxide: a multitasked signaling gas in plants. Mol. Plant 8, 506–520. Dong, Y., Wang, Z., Zhang, J., Liu, S., He, Z., He, M., 2015. Interaction effects of nitric oxideand salicylic acid in alleviating salt stress of Gossypium hirsutum L. Soil Sci. Plant Nutr. 15, 561–573. Ekin, Z., 2005. Resurgence of safflower (Carthamus tinctorius L.) utilization: a global view. Agron 4, 83–87. El-Tayeb, M., 2005. Response of barley grains to the interactive of salinity and salicylic acid. Plant Growth Regul. 45, 215–224. Elhami, B., Zaare-Nahandi, F., Jahanbakhsh-Godehkahriz, S., 2015. Effect of sodium nitroprusside on physiological and biological responses of olive (Olea europaea cv. Conservolia) under water stress. Int. J. Biosci. 6, 148–156. Esra, K., İslek, C., Üstün, A.S., 2010. Effect of cold on protein, proline, phenolic compounds and chlorophyll content of two pepper (Capsicum annuum L.) varieties. Gazi Univ. J. Sci. 23, 1–6. Floryszak-Wieczorek, J., Milczarek, G., Arasimowicz, M., Ciszewski, A., 2006. Do nitric oxide donors mimic endogenous NO related response in plants? Planta 224, 1363–1372. Gebaly, S., 2007. Effect of foliar application of methanol under two levels of irrigation regime on cotton productivity. Egypt. J. Agric. Res. 85, 615–628. Ghadakchiasl, A., Mozafari, A.-A., 2017. Mitigation by sodium nitroprusside of the effects of salinity on the morpho-physiological and biochemical characteristics of Rubus idaeus under in vitro conditions. Physiol. Mol. Biol. Plants 23, 73–83. Ghobadi, M., Taherabadi, S., Ghobadi, M.-E., Mohammadi, G.-R., Jalali-Honarmand, S., 2013. Antioxidant capacity, photosynthetic characteristics and water relations of sunflower (Helianthus annuus L.) cultivars in response to drought stress. Ind. Crop Prod. 50, 29–38. Gunes, A., Inal, A., Alpaslan, M., Eraslan, F., Bagci, E.G., Cicek, N., 2007. Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.) grown under salinity. Plant Physiol. 164, 728–736. Hayat, S., Yadav, S., Alyemeni, M., Ahmad, A., 2014. Effect of sodium nitroprusside on the germination and antioxidant activities of tomato (Lycopersicon esculentum Mill.). Bulg. J. Agric. Sci. 20, 156–160. Jaleel, C.A., Manivannan, P., Wahid, A., Farooq, M., Al-Juburi, H.J., Somasundaram, R., Panneerselvam, R., 2009. Drought stress in plants: a review on morphological characteristics and pigments composition. Int. J. Agric. Biol. 11, 100–105. Jin, J.-W., Xu, Y.-F., Huang, Y.-F., 2010. Protective effect of nitric oxide against arsenic induced oxidative damage in tall fescue leaves. Afr. J. Biotechnol. 9, 1619–1627. Kadioglu, A., Saruhan, N., Sağlam, A., Terzi, R., Acet, T., 2011. Exogenous salicylic acid alleviates effects of long term drought stress and delays leaf rolling by inducing antioxidant system. Plant Growth Regul. 64, 27–37. Khan, M.I.R., Asgher, M., Khan, N.A., 2014. Alleviation of salt induced photosynthesis and growth inhibition by salicylic acid involves glycinebetaine and ethylene in mungbean (Vigna radiata L.). Plant Physiol. Biochem. 80, 67–74. Khan, M.N., Mobin, M., Abbas, Z.K., Siddiqui, M.H., 2017. Nitric oxide induced synthesis of hydrogen sulfide alleviates osmotic stress in wheat seedlings through sustaining antioxidant enzymes, osmolyte accumulation and cysteine homeostasis. Nitric Oxide 68, 91–102. Khan, W., Prithiviraj, B., Smith, D.L., 2003. Photosynthetic responses of corn and soybean to foliar application of salicylates. Plant Physiol. 160, 485–492. Kong, J., Dong, Y., Xu, L., Liu, S., Bai, X., 2014. Effects of foliar application of salicylic acid and nitric oxide in alleviating iron deficiency induced chlorosis of (Arachis hypogaea L.). Bot. Stud. 55, 1–12. Kumar, P., Tewari, R.K., Sharma, P.N., 2010. Sodium nitroprusside mediated alleviation of iron deficiency and modulation of antioxidant responses in maize plants. AoB Plants 2010, 1–12. Lamotte, O., Courtois, C., Dobrowolska, G., Besson, A., Pugin, A., Wendehenne, D., 2006. Mechanisms of nitric oxide induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells. Free Radic. Biol. Med. 40, 1369–1376. Lei, Y., Yin, C., Ren, J., Li, C., 2007. Effect of osmotic stress and sodium nitroprusside pretreatment on proline metabolism of wheat seedlings. Biol. Plant. 51, 386–390.

6