ACTA BOT. CROAT. 77 (1), 2018 45 Acta Bot. Croat. 77 (1), 45–50, 2018 CODEN: ABCRA 25 DOI: 10.2478/botcro-2018-0003 ISSN 0365-0588 eISSN 1847-8476 Salicylic acid-induced germination, biochemical and developmental alterations in rye (Secale cereale L.) Fatma Yanik1, Özlem Aytürk2, Aslihan Çetinbaş-Genç1, Filiz Vardar1* 1 Marmara University, Science and Arts Faculty, Biology Department, Göztepe Campus, 34722 İstanbul, Turkey 2 Maltepe University, Fine and Arts Faculty, Gastronomy and Culinary Department, Marmara Eğitim Köyü, İstanbul, Turkey Abstract – Salicylic acid (SA) is one of the endogenous plant growth regulators that modulate various metabolic and physiological events. To evaluate the exogenous SA-induced germination, biochemical and developmental altera- tions, different concentrations (10, 100, 500 and 1000 µM) of SA were applied to rye (Secale cereale L.) seeds in hy- droponic culture conditions for 15 days. The observations revealed that seed germination and root elongation were stimulated in 10 µM SA treatment, however they were inhibited in higher concentrations (100 and 500 µM) of SA. Furthermore, there was no germination in 1000 µM SA. The analysis of antioxidant enzymes revealed that although superoxide dismutase activity increased, catalase activity decreased in comparison to control. Besides, lipid peroxida- tion and peroxidase activity increased in 10 µM SA, whereas they decreased in higher concentrations. Similarly total chlorophyll content increased in 10 µM SA, but it decreased in 100 and 500 µM SA treatments. Moreover anthocya- nins and carotenoids increased after SA treatment. In conclusion, exogenous SA application causes developmental and biochemical alterations in rye. Key words: antioxidant enzyme, lipid peroxidation, photosynthetic pigments, salicylic acid * Corresponding author, e-mail: filiz.vardar@gmail.com Introduction Agricultural crops and wild flora are faced with a variety of intense environmental stress factors causing considerable economic losses worldwide (Tuzhikov et al. 2011). Plant de- fense is controlled through various type of protein and non- protein signaling molecules. Most phytohormones, such as ethylene, abscisic acid, jasmonic acid and salicylic acid (SA), have important roles as defensive molecules in the signal- ing pathways (Vicente and Plasencia 2011, War et al. 2011). SA is one of the endogenous plant growth regulators that belong to a diverse group of plant phenolics (Pandey et al. 2013). It affects metabolic and physiological events in rela- tion to growth and development in plants. It has long been known that SA regulates seed germination, adventitious root formation, photosynthetic reactions, cellular respiration, thermogenesis, flower formation and anthesis, seed produc- tion and senescence (Raskin 1992, Singh and Usha 2003, Khodary 2004, Vicente and Plasencia 2011). Numerous re- searches indicated that SA content increases under various types of oxidative stress conditions in plants (Larkindale and Knight 2002, War et al. 2011). SA mediates the recognition of pathogens and activation of defense pathways from local to distal infected tissue inducing systemic acquired resistance (An and Mou 2011). It also modulates plant defense under abiotic stress conditions, being an important signaling mol- ecule (Borsani et al. 2001, Muñoz-Sanchez et al. 2013). SA regulates the activity of enzymatic antioxidants such as su- peroxide dismutase (SOD), polyphenol oxidase (PPO) and peroxidase (POD) in response to excessive production of reactive oxygen species (ROS). However, SA inhibits the ac- tivity of some enzymatic antioxidants, such as ascorbate per- oxidase (APX) and catalase (CAT), leading to excess ROS ac- cumulation as signaling molecule (Hayat et al. 2008). Based on the regulatory and defensive role of SA in mul- tiple developmental processes, exogenous application of SA has attracted attention in induction of plant defense against biotic and abiotic stress factors. However, SA controls plant growth and development, depending on the applied dose and species. According to previous research, a broad range of exogenous applied SA doses (10 nM to 10 mM) was effec- tive in different plant species. (Janda et al. 1999, Shakirova et al. 2003, Vicente and Plasencia 2011). It has been reported YANIK F., AYTÜRK Ö., ÇETINBAŞ-GENÇ A., VARDAR F. 46 ACTA BOT. CROAT. 77 (1), 2018 that higher concentrations may cause inhibitory effects on plant growth and development (Hayat et al. 2008). The ob- jective of the study is to investigate the effects of different concentrations of SA on growth, antioxidant enzymes and photosynthetic pigment content in rye (Secale cereale L.), an important crop worldwide. Material and methods The rye (Secale cereale L. cv Aslım 95) seeds were pro- vided from the Bahri Dağdaş International Agricultural Re- search (Konya, Turkey). The surface-sterilized seeds were germinated in Petri dishes with nutrient solution with or without SA (10, 100, 500 and 1000 µM) in a plant growth room set at 23±2 °C temperature, 45-50% relative humidity and a light intensity of 70 µmol (photon) m–2 s–1 (day/night: 16/8). The nutrient solution was a modified version of Hoa- gland’s solution (pH 6.0) including 5 mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 1 mM KH2PO4, 30 µM Fe(III)-EDTA and standard Hoagland micronutrients (Hoagland and Ar- non 1950). All experiments were conducted three times and twenty five seeds were used for each repetition and experi- mental groups. The germinated control and SA treated seeds were count- ed and germination index was calculated. At the end of 15 days primary root length was measured and relative root growth was calculated according to Schildknecht and De Campos Vidal (2002). Hydrogen peroxide (H2O2) content was assayed accord- ing to Junglee et al. (2014). Control and SA treated roots (300 mg) were homogenized with 2 mL of the extraction buffer (0.1% trichloroacetic acid, 1 M KI, 10 mM phosphate saline buffer) and centrifuged at 12000 g for 15 min at 4 °C (Sig- ma 3K18). The supernatants were incubated in total dark- ness for 20 min and then measured at 390 nm, spectropho- tometrically. The standard curve was used to calculate the H2O2 content. Control and SA treated roots (100 mg) were homoge- nized with 1 mL of cold sodium-phosphate buffer (PBS, 50 mM, pH 7.0). Homogenates were centrifuged at 14000 rpm for 20 min at +4 °C. The supernatant was stored in ice for en- zymatic assays. Superoxide dismutase (SOD) activity was de- termined by the method of Cakmak and Marschner (1992). The reaction mixture containing 2 mL of substrate buffer (100 mM pH 7.0 PBS, 2 M Na2CO3, 0.5 M EDTA, 300 mM L- methionin, 7.5 mM nitro blue tetrazolium, 0.2 mM ribofla- vin) and 2 µL of the supernatant was incubated under 15 W fluorescent lamps for 10 min and measured at 560 nm, spec- trophotometrically. Peroxidase (POD) activity was evaluated by the method of Birecka et al. (1973). The reaction mixture containing 1.5 mL of substrate buffer (0.1 M PBS pH 5.8, 5 mM H2O2, 15 mM guaiacol) and 10 µL of enzyme extract was measured immediately for 2 min at 470 nm, spectrophoto- metrically. Catalase (CAT) activity was assayed as described by Cho et al. (2000). The reaction mixture containing 1 mL of substrate buffer (20 mM PBS pH 7.0, 6 mM H2O2) and 25 µL of enzyme extract was measured by the decrease in absor- bance for 2 min at 240 nm, spectrophotometrically. Lipid peroxidation (LPO) was evaluated by determining the concentration of malondialdehyde (MDA) (Cakmak and Horst 1991). Control and SA treated roots (200 mg) were ho- mogenized with 1 mL TCA (0.1%) and centrifuged at 12000 g for 20 min at +4 °C. The reaction mixture containing 1 mL of substrate buffer (0.6% thiobarbituric acid in 20% TCA) and 250 µL of enzyme extract was incubated for 30 min at 95 °C. The mixture was cooled immediately on ice and centrifuged at 12000 g for 10 min. The supernatant was measured at 532 and 600 nm. Total chlorophyll, chlorophyll a, b and carotenoid con- tents were determined according to Arnon (1949). Control and SA treated leaves (0.5 g) were homogenized with 15 mL acetone (80%) and centrifuged at 3000 g for 10 min at +4 °C. The supernatant was measured at 470, 645 and 663 nm, spec- trophotometrically. The anthocyanin content, one of the non-enzymatic an- tioxidants, was assayed according to Rabino and Mancinelli (1986). Control and SA treated leaves (0.5 g) were extracted in 10 mL methanol:HCl (99:1, v/v) and centrifuged at 12000 rpm for 10 min at +4 °C. The supernatant was measured at 530 and 657 nm, spectrophotometrically. Statistical analysis was performed using one way analysis of variance (ANOVA), (SPSS 21.0 software). The significance of the applications was designated at the P < 0.05 level using the Tukey's test. All data presented are means ± SD. Results To determine the dose-dependent effects of salicylic acid (SA), rye seeds were germinated in Hoagland solution with or without of SA (10, 100, 500 and 1000 µM). According to our results the seed germination percentages were 26.67% in control, 41.33% in 10 µM, 22.67% in 100 µM and 17.33% in 500 µM (Fig. 1A). No germination was recorded at the high- est concentration of 1000 µM. The germination percentages revealed that higher concentrations of SA reduced the seed germination; however 10 µM stimulated the germination in comparison to control. Similarly, relative root elongation was decreased at higher concentrations, but it was stimulated at 10 µM SA. Root elongation was 10.28% in control, 13.66% in 10 µM, 5.93% in 100 µM and 3.73% in 500 µM SA after 15 days (Fig. 1B). To assess the oxidative stress after SA application hy- drogen peroxide (H2O2) content and some antioxidant en- zyme activities were evaluated after 15 days. Based on our results, the content of H2O2, generated after univalent reduc- tion of superoxide radicals was reduced by 25.5% in 10 µM SA. However, it was increased by 2.5% in 100 µM and 16.7% in 500 µM SA with regard to control (Fig. 2A). One of the antioxidant enzymes, SOD is responsible for catalyzing the reduction of superoxide anions into H2O2. After SA appli- cation SOD activity increased by 8.1% in 10 µM, 49.3% in 100 µM and 61.9% in 500 µM with respect to control after 15 days (Fig. 2B). According to our results, after SA appli- cation significant reduction was observed in CAT activities suggesting the inhibition of H2O2 breakdown to water (Fig. 3A). The reduction was 60% in 10 µM, 50% in 100 µM and 35% in 500 µM as compared to control. EFFECTS OF SALICYLIC ACID ON SECALE CEREALE ACTA BOT. CROAT. 77 (1), 2018 47 Lipid peroxidation (LPO) was monitored by the malond- ialdehyde (MDA) level. Whereas 500 µM SA application de- creased the MDA content by 46.2%, the lower concentrations increased the MDA content by 53.9% in 10 µM and 7.7% in 100 µM referring to lipid peroxidation (Fig. 3B). Although 10 µM SA application increased POD activity by 56.3%, it was decreased by 31.3% in 100 µM and 37.5% in 500 µM com- pared to untreated control (Fig. 4A). To assess the possible effects of SA on photosynthetic pig- ments, they were quantified after 15 days (Tab. 1). The results revealed that SA application increased chlorophyll a content. Based on our results the most significant increase was observed by 5.3% in 10 µM SA solution. However, chlorophyll b content was decreased dose-dependently. Correlated with chlorophyll b reduction, total chlorophyll decreased in 100 and 500 µM SA applications. Conversely, after 10 µM SA application, total chlorophyll increased by 3%. In addition, the content of carot- enoids, which are lipid soluble antioxidants functioning in oxi- dative stress tolerance, increased in all SA applications. One of the non-enzymatic antioxidants, anthocyanin, in the class of flavonoids increased by 36% in 10 µM, 2.4 fold in 100 µM and 2.9 fold in 500 µM, compared to control (Fig. 4B). Fig. 1. Seed germination (A) and root elongation (B) of rye treated with different concentrations of SA (10, 100 and 500 µM) after 15 days. The data with different letters are significantly different according to Tukey's test at P < 0.05 for independent samples. Results are expressed as mean ± SD. Fig. 2. H2O2 content (A) and SOD activity (B) of rye roots after 15 days of treatment with different concentrations of SA (10, 100 and 500 µM). The data with different letters are significantly different according to Tukey's test at P < 0.05 for independent samples. Results are expressed as mean ± SD. Fig. 3. Catalase (CAT) activity (A) and lipid peroxidation (B) of control and SA-treated (10, 100 and 500 µM) rye roots after 15 days of treatment. The data with different letters are significantly different according to Tukey's test at P < 0.05 for independent samples. Results are expressed as mean ± SD. YANIK F., AYTÜRK Ö., ÇETINBAŞ-GENÇ A., VARDAR F. 48 ACTA BOT. CROAT. 77 (1), 2018 Discussion Salicylic acid (SA), which is one of the phytohormones, has various regulatory roles in plant metabolism (Raskin 1992, Popova et al. 1997). It has been proposed that SA has an important role in bioproductivity, defense and resistance in plants (Hayat and Ahmad 2007). It has been reported that application of SA promoted seed germination, root and shoot growth in soybean, wheat, gloxinia, violet and Bras- sica juncea plants in a dose dependent manner (Gutiérrez- Coronado 1998, Fariduddin et al. 2003, Shakirova et al. 2003, Hayat et al. 2005, Martín-Mex et al. 2015). This situation can be explained by SA treatment stimulating plant growth by stimulating mitotic activity (Shakirova et al. 2003). Al- though most of the studies mentioned that the applied doses between 10–10 and 10–5 µM stimulated growth and develop- ment, the higher doses caused inhibitory effects (Pancheva et al. 1996, Pancheva and Popova 1998). In the presented study 10 µM SA stimulated seed germination and root growth but the higher concentrations (100 and 500 µM) were inhibitory after 15 days, consistently with the previous results. More- over we observed no seed germination in the highest con- centration of 1000 µM in rye. Under appropriate conditions, ROS generation and scav- enging are balanced in the cells. Cellular homeostasis is man- aged by complex signal transduction pathways (Dutilleul et al. 2003). It has been reported that under biotic and abiotic stresses endogenous SA is accumulated and this accumula- tion indicates that in stressed plants SA plays a crucial role as a signaling molecule in the management of cellular redox homeostasis (Apel and Hirt 2004). Oxidative stress induces the generation of ROS such as superoxide anion (O2•−), hy- drogen peroxide (H2O2), and hydroxyl radical (HO•) in plant cells. The ROS scavengers include enzymatic and non-enzy- matic antioxidants operating in the different cellular organ- elles (Noctor and Foyer 1998, Hernandez et al. 2001). H2O2 is generated from reduction of O2•− by superoxide dismutase (SOD) which is one of the key enzymatic antioxidants. Ex- cess H2O2 causes oxidative stress and catalase (CAT) can di- rectly catalyzes its decomposition into O2 and H2O (Quan et al. 2008). As we presented in our study, although 10 µM SA application induced a slight increase in SOD activity, it was increased progressively in 100 and 500 µM SA. Consistent with the SOD activity results, H2O2 levels were increased in 100 and 500 µM SA. Although CAT levels increased depend- ing on the SA dose, all activities remained lower than that of the control suggesting the inhibition of H2O formation. It is widely known that SA restricts CAT activity and stimu- lates an increase in H2O2 level as has been shown (Chen et al. 1993, Janda et al. 2003). There is also evidence for a com- plicated relationship between H2O2 and SA, which stimulate each other (Rao et al. 1997, Dat et al. 2000). Peroxidase (POD) activity is a common response to vari- ous types oxidative stress factors. Numerous research results indicated that SA-controlled reduction in H2O2 levels is relat- ed to elevated POD activities (Wang et al. 2004). Our results revealed that POD activity increased in 10 µM SA, induc- ing the reduction of H2O2, but decreased in higher concen- trations. This situation suggests that higher concentrations may inhibit POD enzyme activity via alterations in enzyme and/or biochemical pathways. On the other hand SOD may control the integrity of membrane structures of the cell cul- Tab. 1. Total chlorophyll, chlorophyll a, b and carotenoid contents (mg mL–1) in rye leaves treated with different concentrations of SA (10, 100 and 500 µM) after 15 days. The data with different letters are significantly different according to Tukey's test at P < 0.05 for independent samples. Values represent means ± SD. Treatment Chlorophyll a (mg mL–1) Chlorophyll b (mg mL–1) Chlorophyll a/b Total chlorophyll (mg mL–1) Total carotenoids (mg mL–1) Control 302.32c±1.04 167.1a±1.07 1.81 469.41ab±1.58 89.53b±0.7 10 µM SA 318.36a±1.09 163.59a±1.28 1.95 481.95a±1.41 95.90ab±0.35 100 µM SA 304.69c±1.04 159.73ab±0.79 1.91 450.30b±1.61 97.42a±0.24 500 µM SA 308.62b±0.53 146.37b±0.89 2.11 454.99b±1.12 94.96ab±0.42 Fig. 4. Peroxidase activity (A) and total anthocyanin (B) of control and SA-treated (10, 100 and 500 µM) rye roots after 15 days of treat- ment. The data with different letters are significantly different according to Tukey's test at P < 0.05 for independent samples. Results are expressed as mean ± SD. EFFECTS OF SALICYLIC ACID ON SECALE CEREALE ACTA BOT. CROAT. 77 (1), 2018 49 minating in the processes of lipid peroxidation (LPO) de- activation (Zenkov et al. 2001). In the present study, LPO increased in 10 µM SA because of slight SOD activity. How- ever, in higher SA concentrations LPO decreased according to the increased SOD activity. Horváth et al. (2007) also in- dicated that exogenous SA application causes a rapid tran- sient increase of ROS including superoxide anions (O2•−). Considering our results, inefficient SOD activity, which is responsible for reduction of O2•− to H2O2, may result O2•− ac- cumulation in 10 µM SA culminating in LPO. A widely known effect of SA is that it raises the photosyn- thetic pigment levels (Khodary 2004). In the present study total chlorophyll content was increased in 10 µM SA, but it was decreased in higher concentrations, suggesting a de- crease in photosynthetic capacity. However total carotenoids were increased, representing close rates in applied concen- trations. Similarly, Pancheva et al. (1996) reported that chlo- rophyll content decreased after different concentrations (100 µM to 1 mM) of SA application in barley leaves. In addition, Moharekar et al. (2003) indicated that different concentra- tions of SA (5, 10, 50, 100 and 200 mg kg–1) activated the synthesis of carotenoids and xanthophylls but reduced the level of total chlorophyll in wheat and moong. According to Szepesi et al. (2009) 10–4 M SA did not cause any reduc- tion in total chlorophyll, but the amount of carotenoids was slightly increased in Solanum lycopersicum. Higher SA con- centrations may decrease the chlorophyll content due to the SA-induced excess ROS accumulation and the consequent inhibition of plant growth and development (Ma et al. 2013). In photosynthetic organisms, carotenoids serve as ROS scav- enger photoprotectants, having a role in ROS scavenging and LPO suppression (Gill and Tuteja 2010). Anthocyanins are flavonoid pigments located in vacuoles responsible for col- oring fruits and flowers (Grotewold 2006). They are consid- ered to be non-enzymatic antioxidants under stress condi- tions (Kovinich et al. 2012). The function of stress-induced anthocyanins is thought to be that of ROS scavengers (Hat- ier and Gould 2009, Agati et al. 2012). Szepesi et al. (2008) indicated SA pre-treatments increased the accumulation of anthocyanins in both the presence and absence of salt stress. According to our results SA application induced an increase in anthocyanin content as well in carotenoids due to SA-in- duced ROS accumulation. Conclusion Some progress in understanding the effects of SA has been obtained under normal and stressful conditions. Al- though the pathway of signal regulation under biotic-abi- otic stress factors in plant tolerance is still complicated, it is known that SA behaves as a signaling molecule, triggering a cascade of protective reactions. According to our results, exogenous SA application causes alterations to germination, antioxidant enzymes and photosynthetic pigment content in rye, depending on the dose. 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