192 1. Introduction Carnation (Dianthus caryophyllus L.) flowers are one of the most important cut flowers cultivated commercially in the world and they are also used as ornamental plants for decorative purposes in orchards and landscapes (Singh et al., 2005). Vase life of cut flowers is an important charac- teristics in crop quality evaluation and it depends on genetic and environmental factors (Seglie et al., 2011). Ethylene, as a gaseous plant hormone, plays a prominent role in acceler- ating senescence phenomena of most plant organs such as fruits, flowers, and floral buds (Yang and Hoffman, 1984). When plants produce ethylene, or are exposed to an external source of ethylene, receptors perceive its presence and the signal is transferred by downstream active genes. Ethylene, by inducing expression of senescence-related genes, affects the physiological characteristics vegetative and reproductive organs resulting in organ abscission and yellowing (Ahmadi et al., 2008). It has been found that exogenous ethylene in some plants like miniature roses, geranium, and begonia has undesirable effects on flower quality and results in accel- erating senescence and decreasing flower life (Ahmadi et al., 2009; Seglie et al., 2011). Application of external ethyl- ene in rose petals causes a considerable increase in ethylene production and activity of ACC-synthase and ACC-oxidase genes (Ma et al., 2006; Ahmadi et al., 2009), although eth- ylene receptor genes are not affected by external ethylene (Ahmadi et al., 2009). Senescence is an oxidative process in which reactive oxygen species and antioxidants play an important role. In general, reactive oxygen species acceler- ate flower senescence by increasing cell membrane perme- ability due to decreasing proteins and nucleic acids result- ing from different protease and nuclease enzyme activities (Barth et al., 2006). Decreasing antioxidant enzyme activity and increasing peroxidation of cell membrane lipids have been indicated as possible reasons of senescence in differ- ent plant species (Buchanan-Wollaston, 1997). In addition, cell death is accelerated in this phase because of a boost in ethylene production during the senescence period (Ebeles et al., 1992). Plants gain from antioxidant mechanisms to Effects of 1-MCP and ethylene on antioxidant enzyme activity and postharvest physio-biochemical characteristics of cut carnation flower cv. Fortune A. Ranjbar, N. Ahmadi (*) Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University, PO Box 14115-336 Tehran, Iran. Key word: Ethylene, peroxidase enzyme, postharvest longevity, senescence, superoxide dismutase. Abstract: Carnation (Dianthus caryophyllus L.) flowers are one of the most important cut flowers in the world. The majority of carnation cultivars are sensitive to ethylene which affects the physiological and biochemical postharvest characteristics of these flowers. Applying inhibitors of biosynthesis and action of ethylene is important factor to protect the display quality and extend postharvest life. In order to evaluate the effects of 1-methylcyclopropene (1-MCP) and eth- ylene on the antioxidant enzyme activity of cut carnation cv. Fortune and subsequently on the extension of vase-life, this experiment was designed as a Completely Randomized Design (CRD) with three replications. Carnation cut flowers were treated with 1-MCP at concentrations of 0, 0.5, 1 and 1.5 µl/l for 24 h and subsequently exposed to ethylene (1 µl/l) for 16 h. Data were analyzed using MSTAT-C statistical software and means were compared based on Least Significant Differ- ences (LSD) test (p< 0.01). Our results showed that 1-MCP treatment had significant effects on vase life and biochemical characteristics like contents of leaf chlorophyll, petal anthocyanin, petal cell membrane stability and antioxidant enzymes activity such as catalase, peroxidase, and superoxide dismutase. The highest vase life and cell membrane stability were appeared in samples treated with 1.5 µl/l 1-MCP which was significantly higher than 0 and 0.5 treatments, although there was no significant difference with 1 µl treatment. The highest chlorophyll and anthocyanin contents were also measured under 1.5 µl/l 1-MCP which was significantly higher than other treatments. The highest and lowest catalase and peroxi- dase activity were related to 1 and 0 µl/l 1-MCP, respectively. The highest and lowest superoxide dismutase activity was observed in 1.5 and 0 µl/l 1-MCP. In conclusion, application of 1-MCP improved and delayed the onset of senescence symptoms resulted in extending the vase life of cut carnation cv. Fortune. Adv. Hort. Sci., 2015 29(4): 192-198 (*) Corresponding author: ahmadin@madares.ac.ir Received for publication 30 June 2015 Accepted for publication 15 August 2015 193 Ahmadi, Effects of 1-MCP and ethylene on antioxidant enzyme activity of cut carnation flower cv. Fortune alleviate the effects of free radicals. These mechanisms in- clude changes in content of defense-related enzymes such as peroxidase, catalase, polyphenol oxidase and other com- pounds like phenols (Staskawicz et al., 1995). Since post- harvest senescence is an important restricting factor in crop presentation and marketability of many cut-flowers, using high confidence methods to delay crop senescence is of great importance. 1-MCP as an anti-ethylene compound has been proved to be effective in inhibiting ethylene response by competing with ethylene for placing on the site of eth- ylene receptors (Seglie et al., 2011; Daneshi Nergi and Ah- madi, 2014). Studies have shown that 1-MCP has inhibited the phenomena of petal fall in geranium, considering that its effectiveness depends on transport conditions, storage tem- perature, and application times (Cameron and Reid, 2001). Studies on cut carnation showed that all concentrations of 1-MCP decreased ethylene production and chlorophyll de- struction was delayed in comparison with control plants (Asil et al., 2013). Black tulip flowers treated with 1-MCP for 8 h showed maximum anthocyanin till the twelfth day of vase life (Chutichudet et al., 2010). Application of 1-MCP on soya plants decreased hydrogen peroxide in comparison with untreated plants as well as production of ethylene and free radicals but increased the activity of antioxidant en- zymes (Djanaguiraman et al., 2011). Considering the role of 1-MCP as an ethylene inhibitor, the purpose of the current study was to evaluate 1-MCP efficacy in extending display-quality ‘Fortune’ carnation flowers. To gain a deep understanding of biochemical characteristics of cut-flowers, enzyme assays were evalu- ated in this experiment. 2. Materials and Methods Carnation cut-flowers of ‘Fortune’ cultivar were har- vested from commercial greenhouses in Pakdasht (Iran) according to standard indexes. Flowers were immediately transferred to the laboratory of post harvest physiology of the Horticulture Department, Faculty of Agriculture, Tar- biat Modares University. Healthy and uniform flowers were selected for the considered treatments. Cut-flowers were placed in the vase solution and treated with 1-MCP (0, 0.5, 1 and 1.5 µl/l) for 24 h in 200 L glass aquarium chambers. Af- ter 1 h of ventilation, the aquarium lids were re-sealed and ethylene was injected inside each chamber using a Hamil- ton syringe to expose cut flowers to 1 µl/l ethylene for 16 h (Daneshi Nergi and Ahmadi, 2014). After termination of the ethylene treatment, the lids of the glass chambers were re- moved and the vases were placed on the lab bench. Experi- ments were run under the following conditions: tempera- ture 20±2°C, relative humidity of 60-65%, light intensity of 15 µmol/m2s-1 with 12 h light and 12 h darkness (Daneshi Nergi and Ahmadi, 2014). Experimentation was conducted in a completely randomized design with three replications and four treatments. Sampling was carried out to evaluate physiological and biochemical characteristics at desired times and data were analysed using MSTAT-C statistical software; means were compared according to the Least Sig- nificant Differences (LSD) test (P<0.01) and graphs were designed using Excel software. Vase life In this study, vase life was considered the time during which cut-flowers can keep their market quality and before senescence symptoms, including bending of petal margins and wilting, appear (Singh, 1994). Cut-flower durability was based on day distance after the end of cut-flower treat- ment till their ornamental value disappeared. Leaf chlorophyll analysis To measure chlorophyll, leaf samples of 0.5 g were ground using a mortar and pestle with liquid nitrogen, dipped in 15 ml 80% acetone in test tubes and centrifuged at 6000 rpm and 4°C. The absorbance of the solutions was read against blank (solvent) at 663 and 646 nm using a spectrophotometer (BIO-RAD) (Richardson et al., 2002). Chlorophyll content was calculated as follows: Chlorophyll a (µg.ml) = 12/5A 663 -2/79A 646 Chlorophyll b (µg.ml) = 21/51A 646 -5/1A 663 Total Chlorophyll (µg.ml) = Chlorophyll a + Chloro- phyll b Petal anthocyanin Petal samples of 200 mg were pulverized in 3 ml 99:1 (v/v) methanol and hydrochloric acid and the obtained ex- tracts were centrifuged at 12000 rpm for 20 min at 4°C. Supernatants were kept at 4°C under darkness for 24 h. Light absorption was then estimated by spectrophotom- eter at 550 nm wavelength and using silence coefficient (=33000 mol2 cm-1) (Krizek et al., 1993). Petal membrane stability index To determine petal membrane stability, two petal sam- ples, each including 200 mg of each replication, were weighted and dipped in 10 ml double distilled water. One sample was placed in 40°C Benmary for 30 min and the other 100°C Benmary for 15 min. After cooling the sample of 40°C Benmary and reaching room temperature, electrical conductivity of the solutions was measured with a conduc- tivity meter and the stability percent of the membrane was determined according Ezhilmathi et al. (2007) as follows: Membrane stability index (percent) = [1-(C1/C2)] × 100 Enzyme assays Peroxidase (POD) enzyme. Peroxidase (POD) was extracted from 200 mg homogenized samples in 25 mM Na-phosphate buffer (pH 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°C. For assay, a mixture con- sisting of 25 mM Na-phosphate buffer (pH 6.1), 28 mM Guaiacol, 5 mM hydrogen peroxide and crude extract was prepared and its absorbance at 470 nm was detected for 1 min, using spectrophotometer (BIO-RAD). Enzyme activ- 194 Adv. Hort. Sci., 2015 29(4): 192-198 ity was expressed as absorption delta of 470 nm per mg protein (Chance and Maehly, 1955). Catalase (CAT) enzyme. Catalase (CAT) was extracted from 200 mg samples homogenized in 25 mM Na-phos- phate buffer (pH 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°C. The supernatant was transferred to 15 ml tubes and referred to enzyme extract. For assay, a mixture consisting of 25 mM Na-phosphate buffer (pH 6.1), 10 mM hydrogen peroxide and crude extract was pre- pared and its absorbance at 240 nm was detected using a spectrophotometer (BIO-RAD). Enzyme activity was de- scribed by measuring the conversion rate of hydrogen per- oxide to water and oxygen molecules, as the decrease of absorbance per time per mg of protein (Cakmak and Horst, 1991). Enzyme activity was expressed as absorption delta of 240 nm per mg protein. All steps of enzyme extraction were performed on ice. Superoxide dismutase (SOD) enzyme. 200 mg of plant tissues were extracted in 50 mM HEPES-KOH buffer (pH 7.8) containing 0.1 mM EDTA. The homogenate was trans- ferred to centrifuge tubes and was centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was transferred to 15 ml tubes and referred to enzyme extract. For assay, a mixture consisting of 50 mM HEPES-KOH (PH 7.8) con- taining 0.1 mM Na-EDTA, 50 mM Na 2 CO 3 (PH 10.2), 12 mM L-methionine, 75 µM Nitro Blue Tetrazolium (NBT), 1µM Riboflavin and crude extract was prepared and en- zymatic extract as a unit of SOD activity was considered as enzymatic amount which resulted in 50% inhibition of NBT in 560 nm (Chance and Maehly, 1955). Reaction mix absorption was measured by spectrophotometer. Total soluble protein was measured using the Bradford (1976) method. Absorption of 1 ml Bradford reagent along with 100 µl enzymatic extract were mixed completely and registered in 595 nm. Protein content was estimated using calibration curve of cow albumin serum (BSA) (Bradford, 1976). 3. Results and Discussion Vase life Vase life is one of the important post harvest characteris- tics of ornamental plants especially for cut carnation, which is highly affected by ethylene. Reduction of unfavorable effects of ethylene is an appropriate method for enhancing postharvest durability of plants or plant organs and com- pounds like 1-MCP are used extensively in order to alleviate the undesired effects of ethylene in horticultural crops. Re- sults showed that the effect of 1-MCP on characteristics of cut carnation cv. Fortune was significant (P<0.01). Increas- ing 1-MCP concentration enhanced vase life of cut flowers so that the longest vase life (11.8 days) was related to 1.5 µl/l 1-MCP which was significantly (P<0.01) higher than control and 0.5 µl/l 1-MCP, while there was no significant (P<0.01) difference with 1 µl/l 1-MCP. By preventing exter- nal ethylene action, treatment with 1-MCP increased vase life (Fig. 1). In accordance with our results, Yamane et al. (2004) showed that 1-MCP increased vase life of cattleya flowers. It seems that 1-MCP prevents ACC-oxidase and ACC synthase expression, subsequently decreasing ethyl- ene production in primary days of treatment with 1-MCP, resulting in increased vase life (Yamane et al., 2004; In et al., 2013; Yang et al., 2013). This is also in agreement with the results of Chutichudet et al. (2010) who reported that 1-MCP protects tulip cut-flower quality by preventing eth- ylene production (Chutichudet et al., 2010). Hence, increas- ing vase life of cut-flowers treated with 1-MCP is related to inhibiting ethylene action and ethylene biosynthesis (Serek et al., 1994; Serek and Sisler, 2001). Membrane stability index 1-MCP treatment preserved membrane stability in car- nation cut flowers. The greatest membrane stability on the seventh day after treatment was related to 1.5 µl/l 1-MCP with no significant (P<0.01) difference toward 1 µl/l, but it was significantly (P<0.01) higher than control and 0.5 µl/l 1-MCP. 1-MCP can prevent membrane degradation by decreasing the lipid peroxidation which is regulated by ethylene (Yuan et al., 2010) (Fig. 2). Since ethylene is the main factor for increasing respiration rate in climacteric crops and causes an acceleration of physical characteristic changes and cell membrane phospholipids degradation, it seems that 1-MCP treatment protects membrane stability by preventing ethylene action. Chlorophyll content Based on the results, the highest chlorophyll content on the seventh day after treatment measured in 1.5 µl/l 1-MCP showed a significant (P<0.01) difference with the other treatments. The effect of 1-MCP on protecting chlorophyll content is a result of ethylene action and consequently in- hibition of ethylene biosynthesis, which is considered the Fig. 1 - Effect of 1-MCP on vase life of carnation cv. Fortune. Data are means with standard errors (n=3). 195 Ahmadi, Effects of 1-MCP and ethylene on antioxidant enzyme activity of cut carnation flower cv. Fortune most important factor of leaf chlorosis in ornamental plants. In accordance with the present results, 1-MCP treatment at all concentrations decreased ethylene biosynthesis which was followed by reduction of chlorophyll destruction com- pared to control plants (Asil et al., 2013) (Fig. 3). Accord- ing to Serek et al. (1998) 1-MCP inhibited leaf chlorosis in chrysanthemum and geranium cv. Isable (Serek et al., 1998). In this case, the effect of 1-MCP was attributed to binding ethylene receptors. A recent study showed that 1- MCP can keep carbon assimilation in a condition of good efficiency and prevent tricarboxylic acid cycle. Under these conditions, by provoking the biosynthesis of gibberellins, the posthar- vest senescence process is retarded which appeared as pre- serving the postharvest quality (Wang et al., 2014). Anthocyanin content Our results showed that the highest anthocyanin con- tent on the seventh day after treatment was related to 1.5 µl/l 1-MCP with significant (P<0.01) differences with the other treatments (Fig. 4). Positive effects of 1-MCP on in- hibition of external ethylene action, delaying senescence and accordingly protecting suitable cell pH were the fac- tors of anthocyanin photosynthesis pigments. Chutichudet et al. (2010) found that Black tulip cut flowers treated with 300 ppb 1-MCP for 8 h had the highest anthocyanin content till the twelfth day of vase life (Chutichudet et al., 2010). Usually, postharvest destruction of anthocyanin pigments is as a result of bracteoles’ membrane function destruction (Jiang and Chen 1995; Jiang et al., 2004). The stability of anthocyanin may be due to the role of 1-MCP, which can decrease membrane destruction of fresh crops (Hershkovitz et al., 2005). Vacuole pH is enhanced during senescence and anthocyanin gets free of color before destruction (Zhang et al., 2001). Furthermore, anthocyanin destruction occurs as a result of polyphenol oxidase activity (Francis, 1989). The preventing from anthocyanins accumulation in tissues could be ascribed to ethylene action, based on research on Arabidopsis thaliana showed negative self-regulation in relationship of ethylene, carbohydrates and anthocyanins. Stress induced by ethylene resulted in reduced absorption of carbohydrates and consequently decreased accumulation of pigments (Das et al., 2011). Antioxidant enzymes The highest activity of catalase was found in flowers treated with 1 µl/l 1-MCP, however it was not significantly (P<0.01) higher than 1.5 µl/l but was significantly (P< 0.01) higher than control and 0.5 µl/l 1-MCP treatments (Fig. 5). The highest activity of peroxidase was detected in 1 µl/l 1-MCP with significant (P<0.01) differences toward the other treatments (Fig. 6). 1-MCP protected antioxidant en- zyme activity by inhibiting ethylene action and biosynthe- Fig. 2 - Effect of 1-MCP on petal membrane stability of carnation cv. Fortune. Data are means with standard errors (n=3). Fig. 3 - Effect of 1-MCP on petal chlorophyll content of carnation cv. Fortune. Data are means with standard errors (n=3). Fig. 4 - Effect of 1-MCP on petal anthocyanin content of carnation cv. Fortune. Data are means with standard errors (n=3). 196 Adv. Hort. Sci., 2015 29(4): 192-198 sis, and subsequently decreased respiration rate. The petal senescence process causes metabolic and physiological changes, which result in the petal death. Senescence begins with the expression of a set of genes related to senescence and it emerges at metabolic level as oxidative processes and often the catabolic processes in senescence increase are irreversible (Buchanan-Wollaston, 1997). In micro- somal membranes of carnation, many superoxides are gen- erated during senescence (Mayak et al., 1983). Here also, increasing free radicals from ethylene stress in plants un- treated with (0 µl/1) 1-MCP damaged to immunity system of antioxidant and reduction of immunity enzymes activ- ity. When treated with different concentrations of 1-MCP, there was more antioxidant enzyme activity because of decreasing effects of external ethylene. Peroxidase has dif- ferent biological functions, such as detoxification of hy- drogen peroxide, lignin biosynthesis, hormonal signaling and response to stress (Gao et al., 2010). Catalase is con- sidered an important biological factor and its major func- tion is in the process of superoxide metabolism by playing role in releasing oxygen and hydrogen peroxide free radi- cals and preventing creation of hydroxyl radicals (Spanou et al., 2012). Superoxide dismutase like Cu-Zn superox- ide dismutase, Mn superoxide dismutase and outside cell superoxide dismutase play a critical role in inhibition of superoxide (Miao and St Clair, 2009). In fact, peroxidase, catalase and superoxide dismutase play roles in protect- ing the metabolism balance of oxygen in plant tissues (Xie et al., 2003). Superoxide causes lipid peroxidation, cell membrane damage and finally senescence; 1-MCP can af- fect enzyme activities, which remove superoxide (Li et al., 2007). In accordance with our obtained results, increasing antioxidant enzyme (catalase, superoxide dismutase and peroxidase) activity of gladiola florets treated with 1-MCP has been reported (Fig. 7). It seems that this treatment de- creases oxidative stresses in cut-flowers (Hassan and Ali, 2014). In other words, activity of these enzymes is a factor for the protection of cells against oxidative stresses (Zhou et al., 2014). It should be mentioned that even if ethylene decreases in response to 1-MCP, the activity of antioxidant enzymes will increase. In addition, a considerable increase in peroxidase, catalase and superoxide dismutase activ- ity was observed in petals of carnation flower cv. Lilacon purple treated with 0.5 µl/l 1-MCP. The 1-MCP treatment Fig. 5 - Effect of 1-MCP on petal catalase enzyme activity of carnation cv. Fortune. Fig. 6 - Effect of 1-MCP on petal peroxidase enzyme of carnation cv. Fortune. Fig. 7 - Effect of 1-MCP on petal superoxide dismutase enzyme of car- nation cv. Fortune. 197 Ahmadi, Effects of 1-MCP and ethylene on antioxidant enzyme activity of cut carnation flower cv. Fortune decreased hydrogen peroxide and superoxide (peroxide anion) compared to control plants (Karimi, 2014). This decreasing may be a result of low ethylene biosynthesis and inhibition of hydrogen peroxide and peroxide anion by peroxide and superoxide dismutase enzymes (Larri- gaudiere et al., 2004). In asparagus, 1-MCP hindered the ethylene signal transduction and resulted in a delay by af- fecting ethylene biosynthesis, and enhancing superoxide dismutase activity (Zhang et al., 2012). In brief, our study has shown that 1-MCP treatment can delay senescence and increase flower vase life by protecting photosynthesis pig- ments and increasing antioxidant enzyme activity. In conclusion, 1-MC prevented the effects of ex- ogenous ethylene and alleviated the stress conditions induced by ethylene in cut carnation flowers. 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