The Effects of Salicylic Acid and Ascorbic Acid on the Antioxidant System and Activity of Superoxide Dismutase Enzyme of Cut Flowers (Rosa Hybrid Acv

The effects of salicylic acid and ascorbic acid on the antioxidant system and activity of superoxide dismutase enzyme of Cut Flowers (Rosa hybrid acv. Black Magic)

Sevana Ghadimian1, 1. M. Sc of Horticaltural Science, Garmsar Branch, Islamic Azad University, Semnan. [email protected]

Elham Danaei2 2. Professor, Dep. of Horticultural Science, Garmsar Branch, Islamic Azad University, Semnan. [email protected]

Abstract To analyze the post-harvest difficulties of cut Black magic roses, the effect of salicylic acid (100, 200ppm) and ascorbic acid (10 and 20 mg/l) with sucrose 3%, on the antioxidant system and on keeping the freshness of this flower, was conducted in a factorial arrangement, carried out in a completely randomized design, with 6 treatment groups and three repetitions, distilled water & sucrose as control, was performed. In this experiment, in addition to the activity of superoxide dismutase enzyme, properties such as vase life, wet weight, dry weight, ionic leakage, rate of solution absorption, petal anthocyanin, petal chlorophyll, were analyzed. Results showed that using the treatments caused the quantitative and qualitative differences to be significant on a 1% statistical level compared to the control group, and the flowers stayed fresh for 7.8 days with treatment of 200 mg/l of salicylic acid versus 4.2 days for the control group. Furthermore, the use of salicylic acid and ascorbic acid caused a reduction of superoxide dismutase enzyme activity in treatment groups versus the control group, due to influencing the phenol cycle and its antioxidant role. The objective of this study is to analyze the effect of using different amounts of salicylic acid and ascorbic acid on keeping the flowers fresh and the activity of antioxidant enzymes in cut roses and determining the optimum concentration of thisArchive material on post-harvest life. of SID

Keywords: Antioxidant system, Ascorbic acid, Post-harvest, Salicylic acid, Vase life

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1- Introduction The life (the length of time a cut flower stays fresh) of cut flowers is one of the most important qualitative factors. Long life of cut flowers has a significant effect on the demand of consumers and the value of cut flowers. Many factors influence the life of a cut flower. Therefore, knowledge of optimal methods of post-harvest care is one of the success factors in the production and export of flowers and decorative plants. Post-harvest physiology research of cut flowers and potted plants, increase our information about the process of aging, prevention of post-harvest damages methods, and fighting oxidative and environmental stresses, and cause the formation of a number of preservative solutions for cut flowers. Most preservative solutions include substances such as sugars, fungicides, bactericides, anti-ethylene material, growth adjusters, and a weak acid. Yet, different species of plant, even different types of plants show different reactions to chemicals. On the other hand, solutions that are used for the preservation of cut flowers should be inexpensive, easy to use, and harmless to the environment. (Vaezi 2011) Roses are important decorative and commercial plants in the word. Roses are of the type: Rosa, genus: Rosacae, and sub-genus: Rosoideae. There are three types of roses. The production of cut flowers is the most important subset of decorative flowers and plants. On average, more than 800 million cut flowers are produced in Iran annually. Roses are one of the most attractive flowers in the world, due to its various shapes and colors. Iran has vast areas of plantation due to its suitable climate and having lots of sunshine. 1-1- Antioxidant and SOD systems Plants use an efficient system of controlling ROS, based on enzyme antioxidants (superoxide dismutase – SOD, Catalase – CAT, ascorbic peroxide – APX) and non-enzyme antioxidants (carotenoids, tocopherol, glutathione, and phenol compounds) for defending against the adverse effects of ROS against important molecules that are necessary for cell structure and its performance (such as fat, proteins, and nucleic acids). However, the catalytic activity of these antioxidant systems can create negative effects on the flowers, through conditions of repeated stress due to biologic or non- biologic elements in very normal (common) conditions (such as storehouses or commercial settings (Qodir et al 2004). SOD speeds (catalyzes) the transformation of two molecules from O2 to O2 and H2O2. On the other hand, the use of ascorbate as an electron donor, APX enzyme reduces the transformation of H2O2 to H2O. The formation of radical hydroxyl by O2 and H2O2 can be controlled by a combination of non-mutagenic reactions by CAT, SOD, and APX enzymes (Tongetat, 2006). Molecular Oxygen is necessary for the life of all thing including plants. However, a type of active oxygen (ROS), which includes the superoxide anion (O2-), radical hydroxyl (OH), radical pre- hydroxyl (O2H) and hydrogen peroxide (H2O2), produce a natural metabolic process in all secondary cells. In general, under various environmental stress conditions, the level of ROS in the plant cells raises which causes oxidative stress. In fact, among stresses, oxidative stress is the cause of cell damage in plants. The level of ROS in plants may increase due to biologic elements (age, pathogen attacks) or non-biologicArchive elements (heat, cold, dryness, salt,of chemicals, SID mechanical damage). This accumulation causes a homeostasis disorder that causes the reduction of oxidation (Shigeoka et al 2002). One of the common attributes among various types of ROS is their ability to produce oxidative stress in proteins, fats, and DNA. Yet, considering its inner-cell concentration, ROS can play a key role as the molecular signal in the defensive and adjusting response of pathogens, however it operates at very low concentrations (Apel & Hirt 2004). ROS responds to stress conditions in two ways: 1) the effect of ROS on various biological molecules, which cause irreversible damage and cause necrosis of the tissue, and in more severe cases, the death of the tissue. (Girotti, 2001). 2) The effect of ROS on expression of different genes and the signal transfer paths regarding defense (Apel & Hirt, 2004). Superoxide dismutase is the ROS radical controlling enzyme in aerobic species. In plants there are three types of SOD based on their active cofactors: Manganese SOD (MnSOD), Copper/Zinc SOD (Cu/Zn SOD), and Iron SOD (FeSOD). During the metabolic oxidative process, ROSs is produced on a controllable level, and plays a key role in the defense of plants. This can be summarized as follows: 1) Strengthening of the cell membrane by structural changes of carbohydrates in the joints, 2) Induction of the genes related to defense coding of inhibitor proteins of gluconase, kinanase, and other

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proteins, and 3) Cellular death in specific parts of the plant (Reilly et al, 2004). The presence of heavy metals in water, such as Lead (Pb) pollution cause oxidative stress which in turn causes damage to the cell and its components such as the chloroplast, and changes in the metabolites concentrations including protein solutions, prolin, glutathione and antioxidant enzymes (Reddy et al, 2005)

1-2- Ascorbic acid Ascorbic acid (vitamin C) is the result of D-glucose metabolism in plants that influence plant growth and development, and play an important role in electron transfer. Smirnoff et al suggest a biosynthesis and identification of some new enzymes. They also reported that the ascorbate synthesized from H.galactose is from the raw internal production of GDP-manose and L-GDP-galactose. Ascorbic acid is also in connection with various biological activities in plants, including enzyme-production elements, antioxidants, and as electron donors or receptors in the plasma membrane or in the chloroplast. Therefore, knowledge of optimal methods of post-harvest care is one of the success factors in the production and export of flowers and decorative plants. Post-harvest physiology research of cut flowers and potted plants, increase our information about the process of aging, prevention of post-harvest damages methods, and fighting oxidative and environmental stresses, and cause the formation of a number of preservative solutions for cut flowers. Most preservative solutions include substances such as sugars, fungicides, bactericides, anti-ethylene material, growth adjusters, and a weak acid. Yet, different species of plant, even different types of plants show different reactions to chemicals. On the other hand, solutions that are used for the preservation of cut flowers should be inexpensive, easy to use, and harmless to the environment. (Vaezi 2011) Roses are important decorative and commercial plants in the word. Roses are of the type: Rosa, genus: Rosacae, and sub- genus: Rosoideae. There are three types of roses. The production of cut flowers is the most important subset of decorative flowers and plants. On average, more than 800 million cut flowers are produced in Iran annually. Roses are one of the most attractive flowers in the world, due to its various shapes and colors. Iran has vast areas of plantation due to its suitable climate and having lots of sunshine.

1-3- Salicylic acid Salicylic acid was the best compound of drugs sold in the world in a period of time was made in 1898 in Germany (Raskin, 1992). However, John Buchner in 1928 isolated salicylic alcohol glycoside from willow bark in Munich, but later named by Rafacle Piria in 1938 as salicylic acid (SA). The word salicylic acid (SA) is rooted in the Latin word "Salix", meaning willow bark, and the reason is that, in all whole plant kingdom (Raskin, 1992), SA plays diverse role in regulating metabolism in plants (PoPova et al., 1997). In terms of chemical structure, SA belongs to a diverse group of plant phenol, which has an aromatic ring with a hydroxyl group or a function of its derivatives. Free SA is a crystalline powder, which can be melted at the temperature 157-1590c. SA is relatively soluble in water and has greater solubility in polar organic solvents and its PH in aqueous solutions is 2.4 (Raskin, 1992). Aspirin is similar to SA, which is the result of a spontaneous hydrolysis AA in aqueous solution (Mitchel etArchive al., 1967). Scientists largely believe of that, SA SID (ortho-hydrobenzic acide) is a natural derivative of cinnamic acid, which is a mediator in the Shikmic acid path, and an effective factor in the synthesis of phenolic compounds. However, there are two possible ways are provided in this direction. Salicylic acid is known to form combinations with the number of molecules glycosylation, or in less by esterification (Popova et al., 1997). Comparison of the effect produced by 22 phenolic compounds shows that SA and ASA are prevented ethylene production. Also, SA is an inhibitor to form ethylene enzyme has dinitrophenol (Leslie and Romina, 1980). Salicylic acid (SA) is an important signaling molecule involved in immune response against biotic and a biotic stresses.

2- Materials and Methods In this study, branch of the Cut Rose flowers, the Black Magic was prepared from Mr. Hussein Banaian's greenhouse with standard conditions in the PAKDASHT city, and was transferred to the laboratory in favorable conditions. This experiment was carried out in October 2012 in the laboratory of Islamic Azad University of Garmsar, as well as Behin azma soil laboratory. During experiment, the

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laboratory temperature was 22 ± 2 ° C, relative humidity, 55 to 75 percent and light cycle was as 12 hours of light and 12 hours of darkness. Branches of the cut flowers to minimize experimental error again in the laboratory were prepared similar in terms of the stem length, and after stem cutting with the length of 40 cm were placed in containers of 500 ml chemical solution. We've disconnected the bottom of the flowers obliquely, so that, firstly, the flowers' height was made uniform. Secondly, if during harvest, the air bubbles were created in the vessels that can impede water movement to end parts of the flower, in so doing, the bubbles were removed and water will easily take its course. The study was carried out to investigate the effect of long-term treatment of salicylic acid in both 100 and 200 ppm levels in combination with 30 mg/lit Sucrose and ascorbic acid in both the 20 and 10 mg levels with 30 mg of sucrose, and 3%distilled water and sucrose was used as control. The experiment was carried out with 6 treatments and three replications and each replication contains 5-demo unit (see Table 1) as factorial experiment in a randomized complete block. Then, the data were first recorded in Excel software, and analysis was performed using the software SPSS and using Duncan's multiple range test, and drawing graphs using Excel software. Assessment and sampling was carried out on days zero, 2, 4 and 6, and evaluated traits include:

Table 1: Abbreviations of the treatments Row Name of treatment Symbol 1 Control of Distilled water Control Dw 2 Sucrose Sucrose 3 Ascorbic acid ppm 10 AS10 4 Ascorbic acid ppm 20 AS20 5 Salicylic acid ppm 100 SA100

6 Salicylic acid ppm 200 SA200

2-1- Life Index The durability of cut flowers, which is one of the most important factors of post-harvest, is calculated as the number of days after harvest, the time to apply chemical treatments to symptoms such as wilting flowers, bending the neck of the flowers, changing color or loss of the petals. The life of cut flowers, which is considered as the number of days after harvest as long as a flower withers, is one of the important characteristics to assess the effect of different treatments, and new varieties and species. In the flowers, the following will be expressed: aging and wilting petals, leaves loss or curvature of the neck of the flowers, full back of petals towards the outer, and change their color that reduces its marketable (October Fereiduni et al., 2009).

2-3- Dry weight Archive of SID Flowers after the end of their life were dried for 96 hours in the oven with a temperature of 62 and then weighed by a digital scale with precision 0.01. By subtracting the weight of the dry weight, the amount of water content can be achieved (Jones et al, 1993).

2-4- Solution absorption Obstruction of the stem vessels is created for various reasons such as entering the air bubbles at the time of cutting stem or microbial growth in solutions because there is sucrose, which leads to reduced water absorption, and ultimately, accelerate the wilting flowers. In this study, the absorbed solution volume was measured by measuring the decrease in volume of the solution in the flask containing flowers, and was expressed as mL (Zamani et al, 2011). To measure this trait, after putting the flowers in the vase solution in graduated cylinder, the mouth of the container will completely block to prevent evaporation of the solution, and reduce the amount of solution in the container is only due to its absorption by flowers. To calculate the absorption solution, the following formula is used:

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(Formula 1) ml.day¯¹.stem¯¹=(VT-1V1)

Where, VT-1 of solution volume is measured in days, and V1 is the volume of the solution the day before (Hetarachchi, 2005).

2-6- Chlorophyll content in Leaves For the measurement of chlorophyll, the Arnon method (1949) was used. Thus, 1 gram of fresh leaf was pulverized in a porcelain mortar with 10 ml of 80% acetone, and optical absorbance of the resulting clear solution after centrifugation was measured by spectrophotometer, at 645 and 663 nm wavelengths, and the finally, chlorophyll content in milligrams per gram of wet tissue was measured by the following formula.

(Formula 3) Chlorophyll a = (12/7*A663) - (2.69*A645) (Formula 4) Chlorophyll b = (4/68 A663) - (22/9 A645 (Formula 5) total chlorophyll (8 / 02A663) - (20/2 A645)

2-7- Ionic leakage in the cell Measurement of ion can be a good indicator in determining the stability of the membrane in water with high EC. Salinity stress in plants leads to oxidative stress, osmotic stress and ion toxicity. To assess the extent of damage to the Ionic leakage membrane, the Ben Hamed et al, 2007 method was used. In this way, 0.5 g of flower petals was put into Falcon and 10 ml of distilled water was added. Then Falcon for 30 minutes were placed in a hot water bath with a temperature of 60 degrees F. and then the electrical conductivity of the sample (EC1) was measured using the EC meter of the Metrhom model (Switzerland). Then, Falcons were autoclaved at 120 ° C for 20 minutes and then cooled to a temperature of 25 degrees, the electrical conductivity of the samples (EC2) was re-measured and Ionic leakage was calculated by the following formula (Kazemi et al, 2012).

(Formula 6) EC1/EC2 X 100:

2-8- Superoxide Dismutase Enzyme In order to extract the superoxide dismutase enzyme (Giannopolitis and Ries, 1997), frozen cells in HEPES-KOH buffer were extracted with pH 7.8, containing EDTA mM 0.1. The obtained homogeneous was centrifuged at the temperature 4° C (× 15000gr for 15 minutes). The supernatant obtained was used to measure the activity of superoxide dismutase. Enzyme extract obtained was used to measure the activity of superoxide dismutase, and the buffer HEPES-KOH (50mM) with 7.8 pH containing EDTA (0.1 Mm), Na2O3 (50 mM) with pH10.2, L - Methionine (NBT, 12mM), (nitro Nitro blue tetrazolium (75mM), riboflavin (Mμ1) were added to it. Archive of SID

3- Results and discussion The results of comparison of data showed that, due to the role of antioxidant ascorbic acid, the flowers aging is delayed, and it led to maintain water absorption, and prevent reduced wet weight. To preserve the life of cut flowers, use of protective solutions that act as antioxidants, can play an important role in delaying flowers aging. Ascorbic acid and salicylic acid, as an antioxidant, while maintaining the phenolic compounds present in the petals will increase the durability of flowers. The results were consistent with the results of J. et al (2006) (see Figure 1 and 2).

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Fig (1) Effect of experimental Factors on water absorption of cut flower

Fig (2) Effect of experimental Factors on fresh weight of cut flower.

Anthocyanin, such as phenolic compounds was reduced with aging the rose flowers. Salicylic acid and ascorbic acid prevent a sharp decline of anthocyanins in the petals of roses compared to control (Figure). Anthocyanins belong to phenolic compounds and phenols are involved in the antioxidant system. It is well knownArchive that, phenols play important roleof in the antioxidant SID defense system by the ascorbate peroxides system. Reducing these compounds causes sensitivity of flowers to oxidative stress, and finally, the petals' aging. Of course, given the role of anthocyanins as the protective compounds against environmental stress and oxidative stress, it can be concluded that an increase or maintain these compounds with protective material can be effective to improve the quality of cut flowers (Figure 3). These results are consistent with the results of Banaei et al (2013).

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Fig (3) Effect of experimental factors on anthocyanin petals of cut flowers.

Carbohydrates evacuation is associated with blackened leaves and necrotic burns due to death of cells in the leaves of cut flowers. Aging leaves of a plant is part of a plant's life, which can be aggravated by stress such as salinity, low light due to metabolic changes such as an increase in the activity of protease, chlorofilas and nucleases, and more. All of these lead to an overall reduction in the amount of protein, RNA, chlorophyll. Salicylic acid and ascorbic acid increases the amount of chlorophyll in the leaves of cut rose flowers (4). These results are consistent with Solgui et al (1392).

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Fig (4) Effect of experimental Factors on chlorophyll index of cut flower.

When the plant is under oxidative stress has special protection mechanisms to deal with these oxidants, including antioxidant molecules and enzymes. Antioxidants are generally divided into three groups, which include membrane compounds and soluble compounds in tocopherol and carotenoids, soluble substances in water such as ascorbate, glutathione, water-soluble, phenolic compounds, anthocyanins and enzymes such as superoxide dismutase, ascorbate peroxidase and glutathione ducats. Phenolic compounds (salicylic acid) and antioxidants (ascorbate) play a major role in the decomposition of free radicals, and reduce the ionic leakage membrane damage and so on. The use of

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ascorbic acid and salicylic acid reduces ionic leakage in cut rose flowers (Figure 5). These results were consistent with the results of Momeni et al (2012).

Fig (5) Effect of experimental Factors on ionic leakage of cut flower.

During the response against pathogens, strengthening the cell walls plays an important role in the defense mechanisms against the penetration of fungal pathogens. In response to defense against pathogen attack, large amounts of ROS will be produced, while reducing the capacity of detoxification, then the accumulation of ROS and activation of the Programmed cell-death occurs. Suppressing removal mechanism of ROS is very important for PCD. H2O2 produced locally and systemically in response to mechanical damage occurs with wounds. Salicylic acid increases the amount of non-enzymatic antioxidants and antioxidant enable Dfayy system that led to the loss of free radicals in the plant. The use of salicylic acid increases the activity of superoxide dismutase cut flowers were roses (Figure 6). These results oriented Lu et al (2011) was adapted. Production of H2O2 occurs locally and systemically in response to mechanical damage with wounds. Salicylic acid increases the amount of non-enzymatic antioxidants and enables antioxidant defense system, which led to the loss of free radicals in the plant. The use of salicylic acid increases superoxide dismutase activity of cut rose flowers (Figure 6). These results were consistent with the results of Lu et al (2011).

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Fig (6) Effect of experimental factors on SOD activity of cut flowers.

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At end of the vase life of rose flowers, the flowers lose their ornamental value that simultaneously, the flower tail is curved, and flower withers. Improving all traits such as wet weight, plant pigments, and enzyme activity leads to improve vase life in treatments compared to control (Figure 7).

Fig (7) Effect of experimental factors on vase life of cut flowers.

4- Conclusion In this study, long-term treatment with acetylsalicylic acid and ascorbic acid, each with two levels, with 3% sucrose was used. 3% sucrose and distilled water as well, were used as control. Traits such as the shelf life of plant, relative wet weight, petal anthocyanins, leaf chlorophyll, cell ionic leakage, dry weight, solution absorption and activity of superoxide dismutase were evaluated. The results showed that, among the six treatments used, Salicylic acid 200-ppm acid with sucrose (3%) more increased the shelf life of plant, and improving quantitative and qualitative characteristics compared to control. Among different levels of salicylic acid used as a continuous treatment, the concentration of 200 ppm and between levels of ascorbic acid used, concentrations of 20 mg showed the greatest effect in improving yield and increasing vase life. The longest length of life among 6 treatments was related to salicylic acid, 200 ppm with 7.8 days, and the lowest lifetime was obtained for control treatment with 4.2 days. In general, treatment of cut flowers with the bestArchive chemical compounds reduces cell ionicof leakage, SID and increases the activity of enzymes, and improves traits such as relative wet weight, petal anthocyanins, solution absorbance, etc. and thus increases the shelf life of rose flowers. Furthermore, the correlation-evaluated traits represent a significant positive or negative relationship between at the level of 1% between all traits, except relative wet weight, dry weight. The correlation between these two traits was significant at the 5% level.

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