Introduction

Drought is an important abiotic stress that limits plant growth in arid and semiarid regions of the world (Li et al. 2013). Plant growth and development under limited water supply could be disrupted by osmotic and oxidative stresses, ion deficit and metabolic disorders (Li et al. 2013). Among other effects, drought may trigger an increased formation of reactive oxygen species (ROS) which can cause lipid peroxidation and membrane damage. ROS such as superoxide radical, hydroxyl radical and hydrogen peroxide are harmful for biological systems, because they oxidize macromolecules such as lipids and proteins and reduce photosynthetic pigments and photosynthesis (Kavas et al. 2013; Ghassemi-Golezani and Nikpour-Rashidabad 2017). Plants could remove ROS by enzymatic and non-enzymatic antioxidants (Mustafavi et al. 2016). Antioxidant enzymes including catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX) and superoxide dismutase (SOD) are very effective in scavenging ROS. SOD produces H2O2 and CAT, APX and POX decompose H2O2 and convert it to O2 and H2O. Phenols and flavonoids are also important scavengers of most types of oxidizing molecules, including singlet oxygen and various free radicals; thereby improving plant resistance to different types of stress (Saeed et al. 2012). Anthocyanin also acts as an antioxidant, counteracting with free radicals and H2O2 (Zamani et al. 2013).

Peroxidation of fatty acids creates by oxidative degradation of lipids. This peroxidation leads to reactive aldehydes production such as malondialdehyde (MDA). MDA content indicates cell membrane injuries and antioxidant potential of plants under stress. Enhancing H2O2 content and subsequently lipid peroxidation under stress diminish membrane stability. Damages of cell membranes under water deficit are also measured by electrolytes leakage (Saruhan et al. 2012).

Soluble sugars and proline, proteins, glycine betaine is compatible osmolytes, which enhance under water deficit and maintain turgor and stabilize cell molecular structure (Shi et al. 2013; Farhangi-Abriz and Ghassemi-Golezani 2018). The accumulation of sugars in cytoplasm protects the membrane proteins and enhances dehydration tolerance (Guo et al. 2018). Compatible solutes protect plants from stress by cellular osmotic adjustment, ROS detoxification, maintenance of membrane integrity and enzymes/protein stabilization. High synthesis of proline from hydrolysis of proteins reduces the effect of water deficit through organic solute accumulation. Proline is a source of energy, carbon, and nitrogen for the recovering tissues. This amino acid also acts as a metal chelator, protects enzymes, activates citric acid (Krebs) cycle, participates in proteins structure, prevents electrolyte leakage (Singh et al. 1973) and regulates osmotic potential (Cvikrova et al. 2013).

Photosynthetic pigments could also be affected by drought stress. Chlorophyll is the main pigment of photosynthesis in plants. Chlorophyll content of leaves reduces as a result of water limitation mainly due to the damage to chloroplasts caused by reactive oxygen species (Farooq et al. 2009). Carotenoids operate as precursors to signaling molecules that influence abiotic stress tolerance (Havaux 2013). These pigments represent a group of lipophilic antioxidants and are able to detoxify various forms of ROS (Bartwal et al. 2013). As an antioxidant, they scavenge 1O2 to protect the photosynthetic system.

Polyamines (PAs) such as spermine (Spm) are small organic polycations found in all living creatures and have important roles in cell cycles, gene expression, signaling, growth and development of plants, and plant resistance to abiotic stresses (Ndayiragije and Lutts 2005). Several studies have shown that PAs such as spermine are involved in the acquisition of drought tolerance in plants (Saruhan et al. 2012). Exogenous PAs improve drought tolerance by increasing antioxidant enzymes activities and photosynthetic efficiency, but this effect strongly depends on PA concentration or type, and stress level (Shi et al. 2013; Shu et al. 2013; Shi and Chan 2014). PAs can also act as free radical scavengers to protect membranes from oxidative damages (Yamaguchi et al. 2007; Parvaiz et al. 2012; Shi and Chan 2014). Spermine plays an important role in drought tolerance of plants (Yamaguchi et al. 2007; Hussain et al. 2011). Comparing the effects of glycinebetaine, salicylic acid, nitrous oxide, brassinosteroid and spermine on rice plants under limited irrigation, foliar spray of spermine was the most effective in stress tolerance of these plants (Farooq et al. 2009).

Safflower (Carthamus tinctorius L.) is produced as oilseed, medicinal and industrial crop. This crop can somewhat tolerate environmental stresses such as salinity and drought (Lovelli et al. 2007). However, the productivity of safflower could be limited under severe drought and salt stresses. Since changes in water-stressed safflower plants in response to exogenous spermine is not clear, this research was laid out to investigate the effects of exogenous spermine on antioxidants, osmolytes, photosynthetic pigments and secondary metabolites of this crop (C. tinctorius L.) under different levels of water supply.

Materials and methods

Experimental conditions

Two preliminary experiments were conducted to select appropriate levels of watering and spermine spray. The final experiment was arranged as factorial based on randomized complete block design with three replications in a glass greenhouse at the University of Tabriz (Iran). Safflower seeds (cv. Goldasht) were obtained from East Azerbaijan Agricultural and Natural Resources Research Center, Tabriz, Iran. These seeds were disinfected by 5% (v/v) sodium-hypochlorite solution for 5 min and then were sufficiently washed with distilled water. In the first experiment, treatments were irrigations up to 100, 80, 60, 40, 20% field capacity after three leaves stage for 14 days. Based on some growth and physiological parameters (data are not shown), 100% and 40% FC were selected as normal watering and drought stress treatments, respectively. In another experiment, plant performance was tested in response to foliar spray of 0, 20, 40, 60, 80, 100 µM spermine, and 0, 40 and 60 µM were selected as appropriate levels of spermine.

After these preliminary experiments, seeds were sown in plastic pots (15 × 15 cm) containing perlite. The pots were irrigated up to 100% FC and then placed in a greenhouse with 25–30 °C, 60% humidity and 16/8 h light/dark conditions. The pots were weighted regularly and water loss was made up by distilled water for the first 7 days, 50% Hogland solution from day 7 to day 14, and thereafter with 100% Hogland solution until three leaves stage of plants. At this stage, the irrigation treatments (100% and 40% FC) were applied, and 2 weeks later spermine (0, 40 and 60 µM) was sprayed on plants. All plants were harvested at 60 days after sowing and physiological and biochemical parameters were measured.

Antioxidant enzymes

0.1 g fresh root and 0.1 g leaf samples were homogenized in ice-cold phosphate-buffered solution (PBS, 50 mM, pH 7), using mortar and pestle. Homogenates were centrifuged at 10,000g for 10 min at 4 °C. The supernatants were used immediately for assessment of the total soluble protein content according to a method introduced by Bradford (1976). Then, the activities of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) were estimated by the following methods:

SOD activity (SOD, EC 1.15.1.1) was assayed via nitro-blue-tetrazolium (NBT) photoreduction inhibition by extracts (Winterbourn et al. 1976). Reaction mixture (3 mL) containing 2.7 mL sodium phosphate solution (1 M, pH 7.8), 100 µL NBT (1.5 mM), 100 µL ethylene diamine tetraacetic acid (EDTA) (1 M) NaCN (0.3 mM), 50 µL of riboflavin and 50 µL enzyme extract. The mixtures were illuminated at light intensity of 5000 Lux for 12 min and the absorbance of the solutions was recorded at 560 nm by a spectrophotometer (Dynamica, Halo-db-20 series, Switzerland). The amount of the enzyme causing 50% protection of NBT photoreduction was considered as one unit, and SOD activity was expressed as U mg−1 protein.

POX activity (POX, EC 1.11.1.7) was determined by recording the increase of absorbance at 470 nm during polymerization of guaicol for 3 min (Obinger et al. 1997). The reaction was initiated by adding H2O2 to the reaction mixture and POD specific activity was calculated, using the extinction coefficient of 26.6 mM−1 cm−1 for guaiacol. One unit of POX activity was considered as the enzyme amount capable of oxidizing 1 µM guaiacol to tetraguaiacol per minute. CAT activity (CAT, EC 1.11.1.6) was estimated by the method of Chance and Mealy (1955). APX activity (APX, EC 1.11.1.1) was measured by following the decrease in absorbance at 290 nm (Boominathan and Doran 2002). The reaction was initiated by adding H2O2 and APX specific activity was calculated using the extinction coefficient of 2.8 mM−1 cm−1 for ascorbic acid and one unit of enzyme activity was considered as the amount of enzyme necessary for the reduction of 1 µM ascorbic acid per minute.

Lipid peroxidation (MDA) and H2O2

The level of membrane lipid peroxidation was determined by measuring malondialdehyde (MDA) (Boominathan and Doran 2002). Plant samples (root and leaf) were homogenized using 3 mL of 0.1 trichlroacetic acid, and the crude extracts were mixed with the same volume of a 0.5% (w/v) tribarbitoric acid solution containing 20% (w/v) trichlroacetic acid. After heating for 30 min and rapid cooling, the absorbance of supernatant was measured at 530 nm. Malondialdehyde concentration was calculated in accordance with the previously prepared standard curve (using 1,1,3,3-tetraethoxy propane) and expressed as µg g−1 dry weight (DW).

The roots and leaves were harvested, immediately frozen in liquid nitrogen, ground and the powder stored at 80 °C. 100 mg of the powder was homogenized with 5 mL of the solution containing 0.25 mL trichloroacetic acid (TCA) (0.1% (w/v)), 1 mL KI (1 M) and 0.5 mL potassium phosphate buffer (10 mM) at 4 °C for 10 min. The homogenate was centrifuged at 12,000g for 15 min at 4 °C. 500 µL of supernatant from each tube were kept at room temperature (about 25 °C) for 15 min. A calibration curve of H2O2 standard solutions was prepared in 0.1% TCA. H2O2 content of the supernatant was estimated by comparing its absorbance at 390 nm with the standard calibration curve (Harinasut et al. 2003).

Total flavonoid, phenol and anthocyanin contents

Measurement of total flavonoids of root and leaf was performed by adding 1.5 mL of 80% methanol, 100 µL of 10% aluminum chloride solution, 100 µL of potassium acetate 1 M, and 2.8 mL of distilled water to 500 µL of each extract. After 40 min, absorbance of mixture was measured at 415 nm. Quercetin was used to construct the calibration curve. The total flavonoid content of the extract was reported in mg of quercetin per gram dry weight (Chang et al. 2002).

To measure the total anthocyanin content in root and leaf, 0.02 g of dry plant tissue was ground with 4 mL of 1% methanol solution of hydrochloric acid in a porcelain mortar. The resulting solution was placed in the refrigerator for 24 h. After centrifuging, the supernatant was removed and absorbance of the solutions was measured at 530 nm and 657 nm. Anthocyanin content of each extract was calculated (Mita et al. 1997) as:

$$ {\text{Anthocyanin }} = \, A_{530} - \, \left( {0.25 \times A_{657} } \right) $$

where A is absorbance at appropriate wavelengths.

Total phenol measurement in root and leaf was performed by adding 2.8 mL of distilled water, 2 mL sodium carbonate 2%, 100 µL of Folin–ciocalteus phenol reagent 50% to 100 µL of each extract. After 30 min, absorbance of mixture was read at 720 nm. Galic acid (GA) was used to construct the standard curve. The total phenol content of the extract was calculated as mg galic acid per g dry weight (Meda et al. 2005).

Soluble and insoluble sugars

Soluble and insoluble sugars were assayed by phenol–sulfuric acid method (Kochert 1978). 5 mL of ethanol (70%) was added to 0.05 g of dry sample and it was kept in a refrigerator for a week. The samples were centrifuged at 10,000g for 15 min. Then, 0.5 mL of the plant extract was made up to 2 mL by adding distilled water, and then 1 mL of phenol (5%) and 5 mL concentrated sulfuric acid were added. The mixture was vortexed and kept for 30 min at room temperature. The sediment was used for determination of insoluble sugars. The absorbance of solution was recorded at 485 nm, and glucose was used to draw a standard curve. The data were expressed as mg g−1 DW.

Soluble protein and free amino acids

The supernatant of extract provided for the measurement of antioxidant enzymes activities in root and leaf was used for determination of the total soluble protein content as described by Bradford (1976). Free amino acids contents were measured by mixing 500 µL ninhydrin reagent and 100 µL of the same extract and placing it in boiling water bath for 4–7 min. After cooling, the absorbance of each sample was recorded at 570 nm and free amino acids contents were calculated using a standard curve prepared by glycine and reported as µg g−1 DW (Hwang and Ederer 1975).

Proline content

Proline was measured according to Bates et al. (1973), using 0.1 g of root or 0.1 g leaf and 2 mL of extraction medium 3% (m/v) sulfu-salicylic acid. Then samples were centrifuged at 2000g for 10 min. Proline was quantified by a spectrophotometer at 520 nm. The proline content was calculated, using a calibration curve, and expressed as µ mole proline g−1 DW.

Photosynthetic pigments

Chlorophylls a, b and total carotenoids of fresh leaves were measured according to Sukran et al. (1998). 0.1 g of fresh leaf was homogenized with 5 mL of 100% acetone and then it was placed in a refrigerator for 24 h. Thereafter, the absorbance of the extract was recorded at 645, 660 and 470 nm. The pigments contents were calculated as mg g−1 DW.

Statistical analyses

Analysis of variance (ANOVA) of the physiological and biochemical data were carried out by SAS (9.2) software. Means were compared by Duncan’s multiple range test at p ≤ 0.05. Figures were drawn by the Excel-2013 software.

Results

Antioxidant enzymes

Water limitation and spermine showed a significant interaction for antioxidant enzymes activities of safflower roots including APX [F (5, 12) = 43.88, p ≤ 0.01], CAT [F (5, 12) = 25.58, p ≤ 0.01], SOD [F (5, 12) = 63.13, p ≤ 0.01], POX [F (5, 12) = 628.75, p ≤ 0.01] and leaves including APX [F (5, 12) = 91.06, p ≤ 0.01], CAT [F (5, 12) = 267.69, p ≤ 0.01], SOD [F (5, 12) = 220.93, p ≤ 0.01], POX [F (5, 12) = 191.43, p ≤ 0.01]. The APX, CAT, SOD, POX activities under drought stress and APX and POX activities under well-watering in roots were significantly decreased as a result of spermine application. In contrast, 60 µM spermine caused a significant increase in CAT and SOD activities in roots with normal water supply. Spermine application (60 µM) significantly reduced POX activity, but enhanced the activities of other enzymes under non-stress condition. However, this treatment was led to a significant decrease in APX and CAT activities and a significant increase in SOD and POX activities of leaves under stressful condition (Fig. 1).

Fig. 1
figure 1

Means ± standard deviations of a ascorbate peroxidase (APX), b catalase (CAT), c superoxide dismutase (SOD), d peroxidase (POX) of roots and leaves of safflower plants in response to water limitation and spermine application. Different letters indicate significant difference at p ≤ 0.05. I1, I2: 100% and 40% field capacity (FC), respectively

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MDA and H2O2 contents

The interaction of water deficit × spermine was also significant for root MDA [F (5, 12) = 350.89, p ≤ 0.01] and H2O2 [F (5, 12) = 4287.68, p ≤ 0.01] and shoot MDA [F (5, 12) = 842.10, p ≤ 0.01], and H2O2 [F (5, 12) = 2196.73, p ≤ 0.01] contents. Application of spermine in stressed and non-stressed plants reduced MDA content of roots and shoots. The lowest amount of MDA in roots and shoots was recorded for plants treated with 60 µM spermine under water deficit. This treatment was led to H2O2 increment in roots, and its decrement in shoots under drought stress. In normal watering, spermine decreased H2O2 content in roots and enhanced it in shoots (Table 1).

Table 1 Means ± standard deviations of MDA and H2O2, total anthocyanin, phenol and flavonoid contents of safflower roots and shoots affected by spermine under different levels of watering
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Total anthocyanin, phenol and flavonoid contents

The interaction of drought stress × spermine was significant for non-enzymatic antioxidants of roots including anthocyanin [F (5, 12) = 227.47, p ≤ 0.01], phenol [F (5, 12) = 60.88, p ≤ 0.01], flavonoid [F (5, 12) = 78.48, p ≤ 0.01], and shoots including anthocyanin [F (5, 12) = 12.38, p ≤ 0.01], phenol [F (5, 12) = 102.25, p ≤ 0.01], and flavonoid [F (5, 12) = 120.38, p ≤ 0.01]. Both rates of spermine increased total anthocyanin, phenol and flavonoid contents in roots under well-watering. The 60 µM spermine also enhanced total phenol and flavonoid contents in roots under limited watering. Spray of 60 µM spermine significantly increased total anthocyanin content in shoots of stressed plants and total flavonoid in non-stressed plants (Table 1).

Soluble and insoluble sugars

The interaction of water deficit × spermine treatment was also significant for root soluble sugars [F (5, 12) = 345.59, p ≤ 0.01], soluble protein [F (5, 12) = 18,934.2, p ≤ 0.01] and proline [F (5, 12) = 614.39, p ≤ 0.01] and shoot soluble sugars [F (5, 12) = 10.51, p ≤ 0.01], soluble protein [F (5, 12) = 24,287.8, p ≤ 0.01] and proline [F (5, 12) = 89.78, p ≤ 0.01] contents. Exogenous application of 40 µM spermine in stressed and non-stressed plants improved soluble sugars of roots. Foliar spray of spermine significantly reduced soluble sugars content in shoots of stressed and non-stressed plants (Table 2). Water deficit and spermine treatment diminished insoluble sugars of shoots (Fig. 2).

Table 2 Means ± standard deviations of osmolytes of safflower roots and shoots affected by different rates of spermine under various levels of watering
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Fig. 2
figure 2

Shoot insoluble sugars content of safflower grown a under levels of 100% and 40% field capacity and b treated by 0, 40 and 60 µM spermine. Means ± standard deviations (n = 3). Different letters indicate significant difference at p ≤ 0.05. DW dried weight

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Protein and free amino acids

Exogenous spermine increased protein content of roots and shoots under water deficit. However, this treatment reduced total free amino acids in shoots of stressed plants. Application of spermine in non-stressed plants significantly reduced protein content in roots and shoots (Table 2).

Proline

In roots, 40 µM spermine enhanced proline content under water deficit. In non-stressed plants, spermine improved proline in roots and reduced it in shoots. Free proline content of shoots under stress was decreased by this treatment (Table 2).

Photosynthetic pigments content

Water deficit and spermine showed a significant interaction on photosynthetic pigments including Chlorophyll a [F (5, 12) = 955.65, p ≤ 0.01], Chlorophyll b [F (5, 12) = 71.60, p ≤ 0.01], and carotenoids [F (5, 12) = 129.59, p ≤ 0.01]. Spermine significantly increased chl a, b and carotenoids contents under water deficit. Chlorophyll a, b and carotenoids were significantly higher in plants treated with 40 µM spermine. In non-stressed plants, photosynthetic pigments were significantly decreased due to spermine treatment (Fig. 3).

Fig. 3
figure 3

The effect of spermine (µM) on a chlorophyll a, b chlorophyll b, c carotenoids of safflower under different levels of watering. Means ± standard deviations (n = 3). Different letters indicate significant difference at p ≤ 0.05. I1, I2: 100% and 40% FC, respectively

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Discussion

Water deficit induces oxidative stress by ROS generation. Plants subjected to drought stress display various defense mechanisms such as stimulation of the antioxidant enzymes system (Kavas et al. 2013). Increasing enzymes activities in roots and leaves under drought stress by ROS scavenging is a defense process that causes plants alleviate some of the harmful effects of drought stress (Salem et al. 2014). Increasing H2O2 of roots under drought stress as a result of spermine application was related with reduction in antioxidant enzymes activities. Enhancing POX and SOD activities under water limitation with 60 µM spermine and increasing CAT activity with 40 µM spermine in leaves caused a reduction in H2O2 content (Table 1; Fig. 1). This could be attributed to free radical-scavenging and membrane protective properties of spermine. Similar results were reported for Cucumis sativus with spermidine under salinity (Duan et al. 2008), dill plants with salicylic acid under salinity (Ghassemi-Golezani and Nikpour-Rashidabad 2017) and cotton plants with putrescine under water deficit (Hanafy Ahmed et al. 2017).

ROS generated during water limitation are highly reactive and alter normal cellular metabolism through oxidative damage to membranes. The extent of oxidative injuries was estimated by the concentration of malondialdehyde. Increasing MDA content of leaves under drought stress was a direct result of lipid peroxidation and membrane damage. Polyamines stimulate the antioxidative mechanisms of defense and reduce the membrane injuries under drought stress (Saruhan et al. 2012). Spermine application diminished these damages in leaves by enhancing the antioxidant enzymes activities and reducing lipid peroxidation in membranes. Improving H2O2 contents in leaves of non-stressed plants by exogenous spermine may be resulted from the catabolism of spermine that produces H2O2.

The concentrations of non-enzymatic antioxidants such as anthocyanins, phenol and flavonoids are influenced by environmental conditions. It was reported that drought stress enhances the amounts of these secondary metabolites in some plants (Ghassemi-Golezani et al. 2013). Increasing anthocyanins in shoots and phenol and flavonoid contents of safflower roots under water deficit (Table 1) suggests that non-enzymatic mechanisms also play the role to overcome the oxidative stress under drought. The anthocyanins compounds of salt stressed red cabbage plants (Hegazi and El-Shraiy 2017) were also enhanced by low water availability. Anthocyanins were considered as an antioxidant pigment and alleviate oxidative stress. Polyamines improved drought tolerance of plants by stimulating the production of phenolic compounds (Hussain et al. 2011). Enhancing phenol and flavonoid contents in roots and anthocyanins content in leaves of drought stressed-plants by spermine spray improves plant resistance to drought by ROS detoxification (Shi et al. 2013). MDA reduction in roots of stressed-plants and in leaves of non-stressed-plants by foliar application of spermine may also be related with enhancing phenols, flavonoids (Table 1) and antioxidant enzymes activities (Fig. 1), respectively. Flavonoid and anthocyanins as antioxidants induce ROS scavenging and inhibit lipid peroxidation (Shi et al. 2013).

Plants can partly protect themselves against water limitation by accumulating of compatible solutes such as soluble protein, solute sugars and proline (Movahhedi Dehnavi et al. 2010). Increasing soluble protein in roots and shoots under water deficit (Table 2) could be associated with accumulation of proteins such as osmotin and dehydrins under low water potential (Gomathi et al. 2013). Accumulation of proteins as nitrogen resources have likely role in osmotic regulation and might be due to de novo synthesis in response to the stress (Razavizadeh et al. 2017).

Rising soluble sugars in shoots (Table 2) under water limitation is related with the increment of invertase activity and decrement of carbohydrates requirement of plants due to growth reduction. Increasing soluble sugars of roots by 40 µM spermine could improve drought tolerance of safflower plants via osmotic adjustment. Decreasing soluble sugars of shoots due to spermine application might be attributed to better water status of polyamine treated plants (Amri and Shahsavar 2010).

Proline is the most common osmolytes that increases in drought stressed-plants and maintains leaf cell turgor (Paul and Roychoudhury 2016). Enhancing shoot proline content under drought stress (Table 2) could be a mechanism for maintaining cell water potential (Movahhedi Dehnavi et al. 2010). Increment of proline could be the result of increasing pyrroline-5-carboxylate synthase (P5C5) activity (Ghassemi-Golezani and Nikpour-Rashidabad 2017). Increment of free proline in roots by 40 µM spermine under water deficit and 40 and 60 µM spermine in non-stressed plants can improve water uptake due to enhancing cell osmolytes. Proline was diminished by application of spermine in safflower shoots due to increasing chlorophyll synthesis, since proline and chlorophyll were synthesized from the same precursor (glutamate). Spermine can act as antioxidant and osmoticum (two main functions of proline) to mitigate some of the reverse effects of stressful conditions (Hanafy Ahmed et al. 2017; Farhangi-Abriz and Ghassemi-Golezani 2018).

Significant reduction of photosynthetic pigments under water deficit (Fig. 3) is mainly the result of damage to chloroplasts caused by reactive oxygen species, and chlorophyll degeneration by chlorophyllase (Paul and Roychoudhury 2016). Deduction in chlorophyll content is identified as a drought response mechanism to minimize the light absorption by chloroplasts (Pastenes et al. 2005). Photosynthetic pigments were increased in stressed-plants by spermine application in favor of decreasing proline content due to similar precursor. Exogenous polyamine improved antioxidant potential of safflower to maintain photosynthetic system (Saha et al. 2015; Mustafavi et al. 2016). In non-stressed plants, enhancing H2O2 (Table 1) reduced leaves chlorophyll and carotenoids contents (Fig. 3) as a result of oxidative stress.

Enhancing root and shoot protein contents by spermine treatment in stressed-plants (Table 2) may be related with prevention of protein degradation by this growth regulator. Polyamines act as a protective for the plasma membrane against stress damage by inhibiting protease activity (Bais and Ravishankar 2002). In addition, they protect plants from stress via stabilizing protein structure and preventing degradation of proteins (Verma and Mishra 2005). PAs covalent binding with proteins by transglutaminase (TGase) helps to stabilize cell structure and function (Campos et al. 2013). Increasing stress tolerance by polyamine was attributed to the increased levels of protein biosynthesis, antioxidant defense reaction, and energy metabolism (Li et al. 2013).

Conclusion for future biology

The beneficial impacts of spermine on safflower plants suggest that it would be worthwhile to examine the capability of this growth regulator and other polyamines in alleviating the harmful effects of environmental stresses on different plant species.