Protective Role Of Salicylic Acid Biology Essay

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This study was carried out to better understand the role of exogenous salicylic acid applied before cold stress to a plant in the cold tolerance mechanism and its time-dependent effect. To that aim, cold-sensitive (Akhisar) and cold-tolerant (Tokak) barley (Hordeum vulgare) cultivars were used. SA (0.1 mM) was applied to 7-d-old barley seedlings growing under control conditions (20/18 °C) and then the seedlings were transferred to cold environment (7/5 °C) at three different times (14, 21 and 28 d). The leaves of 17, 24 and 31-d-old seedlings were harvested and then used in order to determine the activities of apoplastic antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), and ice nucleation activity of apoplastic proteins and electrophoretic patterns of apoplastic polypeptides by SDS-PAGE. Cold treatment caused a regular decrease in the activities of all enzymes in cold-sensitive cultivar; however it increased CAT and POX activities, except for SOD activity, in cold-tolerant cultivar. Exogenous SA applied before cold stress to the leaves of barley increased enzyme activities in both cultivars. Ice nucleation activity increased by cold treatment, especially in 17-d-old seedlings in both cultivars. In addition, SA treatment increased ice nucleation activity in all examined samplings in both cultivars, compared to cold conditions. SA treatment caused accumulation or de novo synthesis of some apoplastic polypeptides separated by SDS-PAGE. The results of the present study show that exogenous SA can improve cold tolerance by regulating the activities of apoplastic antioxidative enzymes, and ice nucleation activity and the patterns of apoplastic polypeptides in the leaves by affecting the parameters on 24 d after application to the barley cultivars.

Additional key words: Antioxidant enzyme, apoplast, cold tolerance, ice nucleation activity, electrophoresis, salicylic acid

Abbreviations: CAT - catalase; POX - peroxidase; SOD - superoxide dismutase; SA - salicylic acid; SDS-PAGE - SDS-polyacrylamide gelelectrophoresis

Introduction

Low temperature is one of the major factors that limit crop production and reduce yield (Janda et al. 2003, Zhang et al. 2011). Plants differ according to their resistance against chilling and freezing temperatures (Levitt 1980). Freezing is lethal to most cellular organisms. Serious effects of cold conditions cause severe deformation on cell membranes and result in cold injury (Mahajan and Tuteja 2005). Dehydration of intracellular environment and physical damage by ice crystals are major causes of freezing injury and death (Levitt 1980). Cold tolerance is the capacity to avoid intracellular ice formation, to withstand extracellular ice formation and to decrease peroxidation of unsaturated fatty acids in phospholipids (Tasgin et al. 2003, Xu et al. 2006). Freezing-tolerant plants suffer from injury only under temperatures at which extracellular ice formation begins (Antikainen 1996). Plants produce several compounds to protect their cells against fatal intracellular and intercellular ice formation. Many overwintering plants accumulate sugars, amino acids and antifreeze compounds including antifreeze proteins in apoplastic (extracellular) region (Livingston and Henson 1998, Yu et al. 2001, Atici and Nalbantoglu 1999, 2003, Tasgin et al. 2003, 2006, Griffith and Yaish 2004, Belintani et al. 2012). Furthermore, cold sensitive plants may suffer from metabolic decomposition when exposed to cold stress, and characteristically exhibit structural injuries (Kacperska 1989, Atici and Nalbantoglu 2003). On the other hand, many plants completing adaptation to cold conditions have evolved a mechanism to enhance their cold tolerance during exposure to low or nonfreezing temperatures in a process known as cold acclimation (Atici and Nalbantoglu 2003, Tasgin et al. 2006, Zhao et al. 2009). Therefore, winter plants can withstand low temperatures that are lethal to many sensitive plants. Effective antioxidant system of cells has an important role in the response of plants to low temperatures, as well as in decreasing cold damage. One of the most important parts of this system is represented by the antioxidant enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6). Antioxidant enzymes allow the elimination of reactive oxygen species (ROS) such as superoxide anion (O2.-) and hydrogen peroxide (H2O2). In the occurrence of cold damage in plants, excessive production of ROS during cold stress has an important role since ROS are highly reactive, and in case of an absence of any protective mechanism, they can seriously disrupt normal metabolism through oxidative damage to membrane lipids, proteins and nucleic acids (Rout and Shaw 2001, Mutlu et al. 2009a, 2009b). In plant cells subjected to stresses, initial events occur mostly in apoplastic space (Vanacker et al. 1998, Atici and Nalbantoglu 2003, Mutlu et al. 2009a). Some researchers studied the effects of environmental stresses on antioxidant system in apoplastic space and suggested that this compartment is important for plant response to biotic and abiotic stresses (Vanacker et al. 1998, Tasgin et al. 2006, Cakmak and Atici 2009; Mutlu et al. 2009a), although intracellular levels of these enzymes are often studied in stress conditions (Ping and Rui 2007, Mutlu et al. 2009b, 2011, Hao et al. 2011, Mallik et al. 2011, Mutlu and Atici 2012).

Examining signal molecules that mediate stress tolerance is an important step in better understanding how plants acclimate to the adverse environments. Some studies indicate that salicylic acid (SA) is an endogenously synthesized hormone for the activation of plant defenses (Hayat and Ahmad 2007). Many researchers examined the role of SA in plant growth and development, flowering, ion uptake, stomatal regulation and photosynthesis (Pancheva et al. 1996, Popova et al. 1997, Uzunova and Popova 2000). Several studies also supported the major role of SA in modulating the plant response to several abiotic and biotic stresses, such as ultraviolet light, drought, salt, temperature, heavy metals and plant pathogenesis (Senaratna et al. 2000, Ananieva et al. 2004, Mahdavian et al. 2008, Kadioglu et al. 2011; Mutlu et al. 2009a, Wen et al. 2008, Guo et al. 2009, Hao et al. 2011, Fu et al. 2011, Saruhan et al. 2012, Mutlu and Atici 2012, Song et al. 2012). Recent studies have reported the effects of SA on cold tolerance. These studies demonstrated that SA treatment increased chilling tolerance in maize (Janda et al. 1999, Horvath et al. 2002), tomato (Ding et al. 2002), banana (Kang et al. 2003), winter wheat (Tasgin et al. 2003, 2006), red globe grape (Li et al. 2005), Brassica juncea (Setia et al. 2006), cucumber (Xia et al. 2007, Lei et al. 2010), radish (Biao 2006), grass (Wang et al. 2009a), rice (Wang et al. 2009b), eggplant (Chen et al. 2011) and barley (Mutlu et al. 2012). However, molecular events involved in SA signaling are not known yet, as well as its effect time in the apoplast of plant following its application.

The present study, therefore, examines the effects of exogenous SA that was applied to plants before cold stress on cold tolerance in two barley cultivars through determining the activities of apoplastic antioxidative enzymes, the ice nucleation activity of apoplastic proteins and the electrophoretic patterns of apoplastic polypeptides by SDS-PAGE. In addition, after SA application, the time elapsing until it was effective on plants was determined according to these parameters.

Materials and methods

Plant material

In this study, cold-tolerant (Tokak) and cold-sensitive (Akhisar) barley (Hordeum vulgare) cultivars were used. Seeds of the plants were provided from The Institute of East Anatolian Agricultural Research, Erzurum, Turkey. Barley seeds were planted in sand in 15-cm pots. They were maintained in a 20/18 °C (day/night),75% relative humidity and a photon flux density of 400 µmol m-2 s-1 photosynthetic active radiation growth chamber with a 12-h photoperiod for 7 d to initiate germination. After 7 d, salicylic acid (SA) solution (0.1 mM) was sprayed on the leaves of plants. Distilled water of the same pH was used to spray the control plant leaves. The plants (with and without SA treatment) were orderly transferred to cold environment (7/5 °C) at three different times (14, 21 and 28 d). Standard nutrient solution (Hoagland) was once added to all pots. The plant leaves were harvested on 17, 24 and 31 d and they were used as research material.

Extraction of apoplastic proteins from leaves

Apoplastic proteins were extracted as described in Hon et al. (1994). Harvested fresh leaves (7 g) were carefully cut with a sharp bistoury into 2 cm lengths, and rinsed in 6 changes of distilled water to remove cellular proteins from the cut ends. At the end of each rinsing, the amount of removed cellular proteins was calculated by measuring at a wavelength of A280. The leaves were then vacuum-infiltrated for 15 min in 20 mM ascorbic acid and 20 mM CaCl2 solution. The leaves were blotted dry and placed vertically in a 20 ml syringe. The syringes were placed in centrifuge tubes. The apoplastic extract was collected from the bottom of the tubes after the leaves were centrifuged at 1500 g for 20 min. Proteins were precipitated from apoplastic extracts by adding 1.5 times the volume of ice-cold MeOH containing 1% HOAc and incubated the samples overnight at -28°C. After centrifugation at 3500 g for 20 min, the protein pellets were washed with 100% ice-cold EtOH and 70% ice-cold EtOH. Contamination of apoplastic extract by cytoplasm constituents, as monitored by the activity of glucose-6-phosphate dehydrogenase was always less than 1% in relation to the catabolic fraction (Mutluet al. 2009a, 2012)

Determination of Enzyme Activities

Apoplastic SOD, CAT and POX enzyme activities in the fractions were measured spectrophotometrically. The SOD activity was estimated by recording the decrease in optical density of nitro-blue tetrazolium dye by the enzyme (Dhindsa et al. 1981). Three milliliters of the reaction mixture contained 2 µM riboflavin, 13 mM methionine, 75 µM nitrobluetetrazolium chloride (NBT), 0.1 mM EDTA, 50 mM phosphate buffer (pH 7.8), 50 mM sodium carbonate and 0.05 ml enzyme fraction. The reaction was started by adding riboflavin solution and placing the tubes under two 30 W fluorescent lamps for 15 min. A complete reaction mixture without enzyme, which gave the maximal color, served as a control. The reaction was stopped by switching off the light and putting the tubes in the dark. A non-irradiated complete reaction mixture served as a blank. The absorbance was recorded at 560 nm, and one unit of enzyme activity was taken as the amount of enzyme that reduced the absorbance reading to 50% in comparison with tubes lacking enzyme. The CAT activity was measured by monitoring the decrease in absorbance at 240 nm in 50 mM phosphate buffer (pH 7.5) containing 20 mM H2O2. One unit of CAT activity was defined as the amount of enzyme that used 1 mol H2O2/min (Upadhyaya et al. 1985). The POX activity was measured by monitoring the increase in absorbance at 470 nm in 50 mM phosphate buffer (pH 5.5) containing 1 mM guaiacol and 0.5 mM H2O2. One unit of POX activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01/min (Mutlu et al. 2009a; 2009b; 2011; 2012).

Determination of ice nucleation activity of apoplastic proteins

Apoplastic proteins obtained from barley leaves were used to determine ice nucleation activity (Atici and Nalbantoglu 1999). The dried protein pellets in the Eppendorf tubes were dissolved in 1 ml HPLC-grade water and the tubes were then positioned in the freezing bath. After equilibration at -1 °C for 30 min, the temperature was lowered stepwise by 0.2 °C intervals. The tubes were allowed to equilibrate at each temperature for 5 min. The tubes were then removed from the freezing bath after the apoplastic protein solution in each tube had been frozen. The freezing temperature was used as a threshold for ice nucleation activity (Mutlu et al. 2012).

Protein electrophoresis of apoplastic proteins

The dried apoplastic protein pellets obtained from the leaves (7 g) were dissolved in equal volumes of sample buffer.Polypeptides of apoplastic proteins were separated in12.5% SDS-polyacrylamide gel (SDS-PAGE) at 110 V (Laemmli1970). The gel was stained with Coomassie brilliant blue (Laemmli1970, Tasgin et al. 2006).

Statistical Analysis

All measurements were performed for 3 times and the means of the values was used. Statistical analysis was performed using a two-way analysis of variance (ANOVA) and means were compared by Duncan´s multiple range test at P<0.05

Results and Discussion

Numerous studies have suggested that enzyme systems located at cell surface or apoplast are the principal sources of superoxide (O2.-) and H2O2 during stress-induced oxidative burst in plant cells (Hernandez et al. 2001, Minibaeva and Gordon 2003,Tasgin et al. 2006, Mutlu et al. 2009a). Although there are many studies on cellular activities of CAT, POX and SOD enzymes in plants applied salicylic acid (SA) or cold (Kang et al. 2003), literature includes a limited number of research papers examining apoplastic activities of antioxidative enzymes under cold stress or SA treatment conditions (Tasgin et al. 2006). Antioxidant enzymes in apoplast of plants under stress such as pathogen attack, ozone, salt, drought were determined to have important roles in regulating stress response (Ranieri et al. 1996, Hernandez et al. 2001, Minibaeva and Gordon 2003, Patykowski and Urbanek 2003, Mutlu et al. 2009a, Saruhan et al. 2012). Recently, Tasgin et al. (2006), using leaves from winter wheat, have reported that exogenous SA treatment could be involved in cold tolerance by regulating apoplastic antioxidant enzyme activities. So, it is pertinent to examine the role of SA in regulating apoplastic antioxidant activity under cold conditions. The present study is the first to examine the time-dependent effect of SA on alleviating cold damage at two barley cultivars (tolerant and sensitive) through determining the activity of apoplastic antioxidant enzymes (SOD, CAT and POX), the ice nucleation activity of apoplastic proteins and the electrophoretic patterns of apoplastic polypeptides.

Effects of cold and cold+SA on ice nucleation activity of apoplastic proteins

An increase in ice nucleation activity value expresses a decrease in freezing temperature, suggesting that the tolerance of plants to cold increases due to delayed freezing temperature in apoplast. In this study, ice nucleation activity in a cold-tolerant barley cultivar was increased by cold treatment in 17-d-old while it was not affected in 22-d-old and it was reduced in 31-d-old seedlings (Fig. 1A). However, the activity of at cold-sensitive barley was also induced by cold in 24-d-old seedlings while it remained unchanged in 17 and 31-d-old seedlings (Fig. 1B). This result may indicate that when the barley plants are exposed to cold, they try to improve adaptation to cold stress by changing the composition of apoplastic proteins. In plants treated with SA before cold stress, the activity in both cultivars was generally increased in 17, 24 and 31-d-old plants when the plants were exposed to cold. The SA treatment showed a similar effect on both barley cultivars and had been more effective in increasing the ice nucleation activity in Tokak than in Akhisar. This result is consistent with the previous study in which SA treatment caused a significant increase in ice nucleation activity in winter wheat leaves under cold (Tasgin et al. 2003). As shown, SA has changed the ice nucleation activity of apoplastic proteins. However, the way SA is involved in is not currently known. The SDS-PAGE of apoplastic proteins from the leaves of barley plants exposed to cold will contribute much to explain these phenomena.

Effects of cold and cold+SA on apoplastic enzyme activities

Cold treatment alone decreased apoplastic SOD activity in both cultivars (tolerant and sensitive) in all samplings (17, 24 and 31-d-old seedlings), compared to respective controls (Fig.1C and D). In addition, SOD activity was gradually increased depending on sampling (especially on 24 and 31 d) in control plants of both species. The effect of cold treatment on apoplastic SOD has not been explained yet. Some researchers have shown that salt treatment decreased apoplastic SOD activity in salt-sensitive pepper (Turhan et al. 2006); however an increased pathogen attack in both resistant and sensitive barley cultivars (Vanacker et al. 1998). Zhang et al. (2009) have also shown that cadmium toxicity induced apoplastic SOD activity in Phaseolus aureus and Viciasativa leaves. SOD is the major scavenger of superoxide (O2ˉ) to form H2O2 and O2, and plays an important role in defense activity against the cellular damage caused by environmental stress (Meloni et al. 2003, Mutlu et al. 2009a, 2009b, 2011). A decrease in SOD activity can induce O2ˉ accumulation excessively produced in plant cells during oxidative stress, suggesting that reduced SOD activity or excessive production of O2ˉ is one of the fundamental factors in metabolic deterioration during cold stress.

Under cold conditions, SA treatment increased (P<0.05) SOD activity in both barley cultivars on 17, 24 and 31 d, compared with respective controls (Fig.1C and D). This result indicates that SA can play a significant role in responding to cold conditions by increasing an impaired activity of SOD through cold stress in barley leaves. Although the effect of exogenous SA treatment on apoplastic SOD under cold condition has not been explained yet, it is reported that under salt stress, the activity of apoplastic SOD is increased by SA treatment (Turhan et al. 2006, Mutlu et al. 2009a), consistent with the results of the present study, which indicate that exogenous SA applied to plant before cold stress increases apoplastic SOD activity, regardless of cultivar during cold stress. The activity of SOD in both cultivars enhanced by SA treatment under cold stress, which is the first step of defense against ROS, causes dismutation from superoxide to H2O2 and hence decreases the risk of hydroxyl radical formation from superoxide via the metal-catalyzed Haber-Weiss-type reaction (Apel and Hirt 2004).

Apoplastic CAT activity increased in 17, 24 and 31-d-old plants when the cold-tolerant barley (Tokak) was exposed to cold, compared to respective controls (Fig. 1E). However, cold treatment reduced CAT activity in cold-sensitive barley (Akhisar) on all the examined days (Fig. 1F). The stimulation of apoplastic CAT activity by biotic and abiotic stresses is a phenomenon that occurs in many kinds of plant species (Gosset et al. 1994, Vanacker et al. 1998, Hernandez et al. 2000, Patykowski and Urbanek 2003, Tasgin et al. 2006, Mutlu et al. 2009a). This data suggests that the activity of apoplastic CAT is significantly increased by cold stress in cold-tolerant genotype of barley while it is decreased in the sensitive one. Likewise, it has been shown that CAT can be involved in the removal of H2O2 in apoplastic environments (Patykowski and Urbanek 2003).

Some researchers suggest that the stress-tolerant genotypes have a better radical scavenging ability (Mutlu et al. 2009a, Mallik et al. 2011, Zhang et al. 2011). Although these results support the findings in the present study, Tasgin et al. (2006) determined that cold treatment caused a decrease in apoplastic CAT activity in winter wheat.

SA treatment before cold conditions, when plants were exposed to cold, significantly (P<0.05) induced apoplastic CAT activity in 17, 24 and 31-d-old seedlings in the both cultivars (Fig. 1E and F). This result shows that exogenous SA treatment increases apoplastic CAT activity, and plays a regulating role in plant apoplast during oxidative burst caused by cold stress. Although Tasgin et al. (2006) determined that SA treatment caused a decrease in apoplastic CAT activity in winter wheat under cold conditions, in the present study, apoplastic CAT activities were induced by SA treatment in 17, 24 and 31-d-old seedlings of the both cultivars. Therefore, such contradictory findings related to exogenous SA treatment can be found in literature. In addition, the adverse findings on the effect of SA on CAT can be also dose-dependent and vary according to plant species (He et al. 2005). The present study concludes that the induced increase of the apoplastic CAT can be considered as an important mechanism in the apoplastic defense strategy. Hence, a significant increase in the apoplastic activity of CAT can be observed in only low SA concentration (0.1 mM) in the both cultivars grown under cold conditions. In an inconvenient concentration, SA, itself, can also be a stress factor. On the other hand, SA could induce apoplastic CAT activity without a tolerant degree of varieties of barley, which can be suggested as an important finding, as well.

The present study also determined apoplastic POX activity, one of the important antioxidant enzymes in plant response to stress tolerance. It is a well-known fact that POX protects cells against the damaging effects of H2O2 during an oxidative-burst response under stress conditions (Levine et al., 1994). POX is also the primary H2O2-scavenging enzyme that detoxifies H2O2 in the apoplast of plant cells. In this study, cold treatment alone stimulated apoplastic POX activity in tolerant cultivar in all examined samplings while generally decreased (P<0.05) at the same days in sensitive cultivar, compared to respective controls (Fig. 1G and H), which suggests that apoplastic POX activity can be also affected by cold stress. In addition, in cold-sensitive cultivar, activities of apoplastic POX were coordinated with apoplastic SOD and CAT induced at the same level in 17, 24 and 31-d-old plants (Fig. 1D, F and H). In our study, the increase in POX activity obviously suggests that the cold-resistant varieties of barley can better scavenge the H2O2 excessively produced during cold stress. Some studies have shown that stress factors such as pathogen attack (Vanacker et al. 1998) and cadmium toxicity (Zhang et al. 2009) increased apoplastic POX activity in leaves of barley, Phaseolus aureus and Vicia sativa. Cold stress also results in higher POX activity in apoplastic area of winter wheat leaves (Tasgin et al. 2006).

SA application before cold conditions, when the plants were exposed to cold, significantly (P<0.05) increased apoplastic POX activity in the leaves of both cultivars, compared to respective controls (Fig. 1G and H). This result shows that exogenous SA treatment contributes to regulating cold stress tolerance by stimulating apoplastic POX activity in barley exposed to cold conditions. It has been reported that exogenous SA treatment results in a higher activity of apoplastic POX in wheat plants under cold stress (Tasgin et al. 2006). Exogenous SA treatment can undertake a significant role in regulating the response to cold stress by both barley cultivars through regulating POX activity. However, the regulatory effect of SA is observed to be more effective in cold-sensitive cultivar in which POX activity was decreased by only cold (Fig. 1G and H). SA is also known to be effective in the processes related to the biosynthesis of some substances such as lignin and suberin which strengthen cell wall by increasing the POX activity (Sakhabutdinova et al. 2004).

Effects of cold and cold+SA on the pattern of apoplastic proteins by SDS-PAGE

The evaluation of apoplastic proteins was investigated by SDs-PAGE. Apoplastic polypeptides from plants under control, cold and cold+SA conditions were separated according to standards. The molecular masses of apoplastic polypeptides were determined to be in the sizes ranging between 16 and 50 kDa (Fig. 2A and B). Cold treatment increased the accumulation of 16-kDa polypeptides in 24-d-old seedlings of cold-tolerant cultivar, but it caused the accumulation of 14, 16, 20 and 29-kDa polypeptides in cold-sensitive cultivar (Fig. 2A and B). It can be seen that the pattern of apoplastic polypeptides is affected by cold treatment in both varieties. Several researchers also determined that polypeptides of low molecular mass (from 15 to 32 kDa) were accumulated to high levels in leaf apoplast during the cold acclimation of winter rye (Marentes et al. 1993) and spruces (Jarzabek et al. 2009). As an important mechanism of adaptation to cold, the accumulation of these polypeptides in leaf apoplast may play a critical role in improving the freezing tolerance in plants. These proteins are probably the ones that increase cold tolerance by impeding freezing point in apoplastic space during cold stress. SA treatment causes the accumulation of polypeptide of 16 kDa in 24-d-old plants (Fig. 2A and B), and caused the synthesis of a new polypeptide with an approximately 24-kDa size in cold-sensitive cultivar and it increased accumulation of polypeptides of 14, 16 and 29-kDa size (Fig. 2B). Similar effects of SA treatment in plants exposed to cold stress were observed in several researches. For example, Antikainen and Griffith (1997), who examined winter rye, determined that apoplastic antifreeze proteins were highly correlated with frost tolerance, and Tasgin et al. (2003), who examined winter wheat, also determined that SA treatment increased accumulation of apoplastic proteins and decreased freezing injury in leaves.

Freezing is known to take place in the apoplastic space of plant leaves since the protein concentration in intracellular area is sufficient to prevent freezing which causes a significant irreversible damage to cell membranes. Therefore, the freezing occurring in apoplast should be managed. Proteins are important elements that impede freezing point and regulate the formation of ice in apoplast (Yu et al. 2001, Griffith and Yaish 2004, Atici and Nalbantoglu 2003). In the present study, exogenous SA treatment applied before cold stress to the leaves of barley increased ice nucleation activity of apoplastic proteins and the accumulation of some apoplastic polypeptides, and also caused a de novo synthesis of some of them. Exogenous SA applied before cold stress can be suggested to improve cold tolerance by regulating the mechanisms in the apoplast of two barley cultivars. It is evaluated as an important result that SA can make this effect in the cold-sensitive variety of barley.

In conclusion, cold treatment caused a regular decrease in all the apoplastic antioxidant enzymes (SOD, CAT and POX) in the cold-sensitive cultivar of barley, while it increased CAT and POX activities except for SOD activity in the cold-tolerant cultivar. Exogenous SA applied to the sensitive and tolerant cultivar of barley before the exposure to cold stress increased the activities of the apoplastic antioxidant enzymes. SA treatment increased the ice nucleation activity of apoplastic proteins, and caused the accumulation or a de novo synthesis of some apoplastic polypeptides separated by SDS-PAGE. In addition, SA treatment was determined to be effective during 24 d in the regulation of the examined parameters after the application to the barley cultivars. It can be suggested that exogenous SA treatment can play an ameliorating role in cold tolerance by regulating the activities of apoplastic enzymes, ice nucleation activity of apoplastic proteins and the patterns of apoplastic polypeptides in both cold-sensitive and cold-tolerant cultivars of barley.

Acknowledgements

This work was supported by TUBITAK Grant No: TBAG- (106T582). This research was part of a Ph.D thesis accomplished by Salih Mutlu at Atatürk University, Graduate School of Natural and Applied Sciences.

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