Impaginato 421 Adv. Hort. Sci., 2018 32(3): 421-431 DOI: 10.13128/ahs-23361 Biochemical, physiological changes and antioxidant responses of cut gladiolus flower ‘White Prosperity’ induced by nitric oxide H. Kazemzadeh-Beneh 1 (*), D. Samsampour 1, S. Zarbakhsh 2 1 Department of Horticulture Science, Faculty of Agriculture and Natural Resources, University of Hormozgan, Bandar Abbas, Iran. 2 Department of Horticulture Science, Plant Breeding and Biotechnology, Faculty of Agriculture, Shiraz University, Shiraz, Iran. Key words: anthocyanin, catalase, cut gladiolus flower, enzymatic antioxidant system, nitric oxide (NO), sodium nitroprusside (SNP) Abstract: Sodium nitroprusside (SNP), as nitric oxide (NO) donor, has been con- sidered by postharvest researchers as one of the best option for slowing the processes controlling senescence in cut flowers. Here, we investigate the role of NO on postharvest physiology and vase life of the Gladiolus grandiflorus cv. White Prosperity. Vase life markedly extended by SNP at 150 μM from 3 day to 7.33 day and thus those inducer effects were dose- and time-dependent. SNP at 125 μM interdependent on vase life time period was observed to be the optimal dose for improving of relative fresh weight (RFW), peroxidase (POD), and total monomeric anthocyanin (TMA) in cut flowers. Supplementing vase solution with SNP indicated significant increase in water uptake of cut flowers and consequently protected to decline in RFW due to alleviate water losses stress. SNP was maintained the level of total soluble protein, lipid peroxidation, and POD, whereas it enhanced the level of catalase (CAT) and TMA in flower petals. Summary of our results revealed that SNP exogenous prolongs vase life via maintaining protein degrade, scavenging free radical in term of anthocyanin and enzymes antioxidant, decreasing polyphenol oxidase, inhibiting lipid perox- idation, and improving membrane stability in ‘White Prosperity’ cut flowers. 1. Introduction Floriculture is an emerging and fast expanding globalized market and subsequently studies on postharvest handling of cut flowers occupy a fun- damental position (Gul and Tahir, 2013). Therefore, the postharvest longevity of flowers have a vital importance in evaluating the value of the each horticulture plant. This aspect can be particularly hold good with cut flowers and it is a necessity for extended handling and transportation periods. Cut flowers are greatly perishable, and consequently they have short vase life and also are exposed to early senescence processing, which restricts efficient marketing of economically significant ornamental plants (*) Corresponding author: kazemzadehhashem@yahoo.com Citation: KAZEMZADEH-BENEH H., SAMSAMPOUR D., ZAR- BAKHSH S., 2018 - Biochemical, physiological changes and antioxidant responses of cut gladio- lus flower ‘White Prosperity’ induced by nitric oxide. - Adv. Hort. Sci., 32(3): 421-431 Copyright: © 2018 Kazemzadeh-Beneh H., Samsampour D., Zarbakhsh S. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 6 June 2018 Accepted for publication 16 September 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(3): 421-431 422 (Nasibi et al., 2014). However, postharvest senes- cence is a major restriction to the marketing of many species of cut flowers and so much appreciable efforts have been dedicated to developing posthar- vest treatments to extend the marketing period or increasing postharvest longevity (Vajari and Nalousi, 2013). Sodium nitroprusside (SNP), as donor nitric oxide (NO, is one of the postharvest treatments which recently using from it for improving posthar- vest life of horticulture crops has exceptionally increased. Postharvest application of SNP has been shown to be effective in extending the postharvest life of a range of flowers, fruits and vegetables when applied as a short term fumigation treatment at low concentrations (Wills et al., 2000). NO is a short-lived bioactive molecule, which is considered to function as prooxidant as well as antioxidant in plants. NO molecule is now documented as an important signal- ing molecule and reported to be involved in various key physiological processes such as plant defense mechanism, abiotic stress resistance, germination, stimulate antioxidant compounds, decrease lipid per- oxidation, growth and development of plants etc. (Zhao et al., 2004). Furthermore, it was also revealed that plant response to such stress or like drought, high or low temperature, salinity, heavy metals and oxidative stress derived from reactive oxygen species (ROS), is moderate by NO (Mandal and Gupta, 2014 ). NO is recognized as a biological messenger in plants and it has been proved that NO is effective for increase the vase life of cut flowers because it can be may play role as anit-ethylene synthesized from wounded or non-wounded organ (Abasi, 2014). Liao et al. (2009) reported that NO may act as an antago- nist of ethylene in cut rose flowers senescence. Optimum SNP levels could postponement the climac- teric phase of many tropical fruits and elongate the post-harvest shelf life of a wide range of horticultural crops by inhibiting ripening and senescence (Singh et al., 2013). Gladiolus is one of the four famous cut flowers in the world (Bai et al., 2009). Gladiolus cut flowers have extremely used to decorate graves and cele- brate major life events in Iran. Likewise, the longevity of cut flowers is one of the main challenges of florists today. First data concerning about the effect of SNP on differential activity of antioxidants and expression of SAGs (senescence associated genes) in relation to vase life of gladiolus cut flowers (Gladiolus grandiflo- ra cv. Snow Princess) has been reported by Dwivedi et al. (2016). Finding of their study suggested that the application of SNP increases vase life by increas- ing the scavenging mechanism of reactive oxygen species (ROS) in terms of antioxidants activity, mem- brane stability and down-regulation of GgCyP1 gene expression in gladiolus cut flowers. Under condition in plants subjected to SNP, not only the responses of various genotypes or cultivars to SNP may be multi- response, but also the responses rely on dose-and cultivar-dependent, physiological growth state, and environmental factors status. The same trend has been stated by Naing et al. (2017) who found that SNP promoted the vase life of the cut gerbera flow- ers via a delay in the time to stem bending; however, all three gerbera cultivars responded to SNP and the effects were found to be dose- and cultivar-depen- dent. In the previous study, it has been demonstrat- ed that the SNP dose that was best for one cultivar was not suitable for another; thus, variation in the o p t i m a l d o s e o f S N P a m o n g c u l t i v a r s f o r t h e enhancement of their vase life could result from dif- ferences in their genetic background (Naing et al., 2017). Hence, whether SNP participates in improving of cut flowers of White Prosperity cultivar has not been yet reconnoitered. However, the purpose of the pre- sent study was to evaluate the effect induced by nitric oxide donor namely, SNP, on the enzymatic antioxidant activity, biochemical and physiological processes of cut gladiolus (Gladiolus grandiflorus cv. White Prosperity) flowers in order to extend their vase life and postharvest shelf-life. 2. Materials and Methods Plant material and SNP treatments Cut flowers used in the experiment were G. gran- diflorus cv. White Prosperity. Cut gladiolus flowers were obtained from a commercial grower presented in Mahallat city, as famous central commercial pro- duction of ornamental plant, in Iran at normal har- vest maturity and transferred immediately to labora- tory of the Postharvest Physiology and Technology R e s e a r c h , F a c u l t y o f A g r i c u l t u r e a n d N a t u r a l Resources, Hormozgan University at Jun, 2017 and the experiments were established on the same day. Flowers stems ends were recut under tap water to eliminate air emboli, to inhibit vascular blockage, and to trim to a uniform length of 70 cm. Stock solutions of SNP (Enzo Life Sciences) were prepared following the manufacturer’s instructions. Uniform cut flowers Kazemzadeh-Beneh et al. - Postharvest physiology of cut gladiolus flower “White prosperity” 423 were placed in holding solutions, that containing of SNP, Na2 [Fe (CN) 5 NO]. 2H2O (Sigma-Aldrich), as NO donor (0, 25, 50, 75, 100, 125 and 150 μM) plus 3% sucrose as carbohydrate supplement. For control set, flowers were dipped in distilled water plus 3% sucrose. Finally, the flowers stems were placed in 500 ml bottles with 250 ml of each mentioned solu- tions containing different concentrations of the SNP solutions + 3% sucrose and they were maintained at a temperature of 23±3°C, 60±5% relative humidity and under a 12 h photoperiod using cool-white fluo- rescent lamps (24 μmol m-2 s-1 irradiance) during experimental period. There were three bottles (21 flowers) per treatment and the experiment was done seven treatments. To escape from photodegradation of SNP (release of a nitrosyl ligand and a cyanide ion), the bottles were shielded with black nylons. SNP treatment was applied as a continuous treatment and flower stems were kept in solutions till the end of vase life. Vase life and water uptake The vase life was determined based on wilting of more than one-third of the petals of flower and vase life termination of each floret was considered as soon as the first symptom of wilting was observed. Indeed, it was defined as the number of days in vase life required for one-third of the florets of each spike to lose its ornamental value (lost turgor and wilted). Water uptake was measured by periodically weight- ing the vase of a control bottle without cut flowers and bottles containing flowers. Finally, vase water u p t a k e w a s d e t e r m i n e d u s i n g t h e f o r m u l a (Rezvanypour and Osfoori, 2011): Water uptake (ml day-1 g-1 fresh weight) = (St-1-St)/Wt Where St= solution weight (g) at = days 1, 4, 8 and St-1= solution weight (g) on the preceding day, and Wt = fresh weight of the cut flower (g) on t days. Number of opened, unopened florets and relative fresh weight O n e a c h s p i k e , t h e n u m b e r o f o p e n e d a n d unopened florets was recorded from the beginning of the experiment to until 20 days after SNP treat- ments. The fresh weight was measured every 4 days and relative fresh weight (RFW) of cut flowers was calculated by the following equation: RFW (%) = (Wt/W t=1 ) × 100 where Wt = weight of cut flowers (g) at t = days 1, 4, 8 and Wt=1 = the initial fresh weight of the same cut flower (g) on day 1 (Rezvanypour and Osfoori, 2011). Antioxidant Enzyme assays Antioxidant enzyme activities were determined in the third floret from the base of spike at three time points (days 1, 4, and 8). The 100 mg of floret tissue from controls and SNP treatments were removed, were homogenized with mortar and pestle in 1 mL 50 mM EPPS buffer (pH 7.8) containing 0.2 mM EDTA and 2% PVP, and were ice-covered for the analysis of antioxidant activity. The homogenates were cen- trifuged at 4°C for 20 min at 12 000×g and the obtain- ing supernatants were used to evaluate of antioxi- dant enzyme activities. Catalase (CAT) activity was assayed as described by Chance and Mahly (1995) as follows: the assay reaction mixture of CAT contained 50 mM phosphate buffer (pH 7.8), 15 mM H2O2, and crude enzyme. The decomposition of H2O2 was fol- lowed at 240 nm (E = 39.4 mM-1 cm-1). Absorbance values were quantified using standard curve generat- ed from known concentrations of H2O2. For the mea- surement of peroxidase (POD) activity, the reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM guaiacol, 5 mM H2O2 and enzyme. The reac- tion was started by adding 300 μl of H2O2 (0.03%). The POD activity was determined by the increase in absorbance at 470 nm due to guaiacol oxidation (E = 26.6 mM-1 cm-1) (Chance and Mahly, 1995). The polyphenol oxidase (PPO) activity was assayed in 2.8 mL of reaction mixture comprised 2.5 mL of 50 mM potassium phosphate buffer (pH 7.8), 0.3 mL sub- strate containing 0.2 mL pyrogallol and 0.1 mL crude enzyme (Kar and Mishra, 1976). The reaction mixture was mixed and the PPO activity was determined in absorbance at 420 nm (6.2 mM-1 cm-1). It’s to be remembered that the blank cuvette consisted of 3.0 mL potassium phosphate buffer (pH 7.8). The results of antioxidant enzymes activitie was expressed as units (U) per mg FW. Anthocyanin content assay Petals were cut from controls and SNP treatments of at three time points (days 1, 4, and 8) and were frozen for the analysis of total monomeric antho- cyanin (TMA). TMA content in petals extract was determined by the pH-differential method based on two buffer system described previously by Giusti and Wrolstad (2005). To measure the absorbance at pH 1.0 and 4.5, the samples were diluted 2 times with pH 1.0 potassium chloride buffer (0.025 M) and pH 4.5 sodium acetate buffer (0.4 M), respectively. Therefore, the TMA content analyses of prepared mixtures were performed following the methods of Giusti and Wrolstad (2005). Adv. Hort. Sci., 2018 32(3): 421-431 424 Lipid peroxidation The level of lipid peroxidation in petals tissue was measured by determination of malondialdehyde (MDA), which is recognized to be breakdown prod- ucts of lipid peroxidation, at the end of time points (days 8). The MDA content was determined with the thiobarbituric acid (TBA) reaction. Temporarily, 0.2 g of sample tissue was homogenized in 5 ml 0.1% TCA. The homogenate was centrifuged at 10000 g for 5 min. 4 ml of 20% TCA containing 0.5% TBA were added to 1 ml aliquot of the obtained supernatant. The mixture was heated at 95 ºC for 15 min and cooled immediately on ice. The absorbance was mea- sured at 532 nm by a spectrophotometer. The value for the non-specific absorption at 600 nm was sub- tracted from the above value. The level of lipid per- oxidation was expressed as mmol of MDA formed using an extinction coefficient of 155 mmol-1 cm-1 (Heath and Packer, 1968). Total soluble proteins Total soluble proteins (TSP) content of petals at the three time point (days 1, 4, and 8) was deter- mined according to the method of Bradford (1976) using Bovine serum albumin as standard. Statistical analysis The experiment was carried out in completely randomized design (CRD) with three replications. Three flowers stems were used for each replication and thus, the experiment was done with seven treat- ments and three replication per treatment. The non- normalize date of the total soluble protein, POD enzyme activity and RFW of cut flowers were normal- ized with kurtosis and skewness test; so, their trans- formed date used for analyzing. Data were statistical- ly analyzed using analysis of variance (ANOVA) in SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). Correlations among the evaluated parameters were analyzed using Pearson’s correlations (p<0.05 and p<0.01). Mean comparisons to identify signifi- cant differences between treatments were per- formed using Least Significant Difference (LSD) at the p<0.01 or 0.01 level of probability. 3. Results Vase Life, RFW and water uptake Application of SNP markedly enhanced the time to vase life for White Prosperity cultivar (p < 0.01). Results showed that the bottle solution containing SNP + sucrose, significantly increased the vase life of cut flowers compared to the control solution (dis- tilled water), as maximum vase life with higher con- centration of SNP treatments was verified near the end of storage (Table 1). However, it positive impacts on increasing vase life was dose-dependent: 150 and 125 μM were displayed to be the best concentration for vase life (7.33 and 5.66 days) of ‘White Prosperity’, respectively, whereas the other concentrations lower than the 125 μM did not markedly influenced vase life as compared to control (p<0.01). Generally, based on the results of vase life, ‘White Prosperity’ exhibited a longer vase life (4.33 days) when exposed to 150 μM NO as compared to control (3 days). The prolonged vase life in SNP-treated cut flowers were approximately associated with increasing in floral opening of cut flower by 150 μM treatment (Table 1). A d i r e c t s i g n i f i c a n t r e l a t i o n s h i p w a s d e t e c t e d between SNP and floral opening (%); however, increasing in SNP concentration resulted in increasing in floral opening percentage. Statistically, the floral abscission and un-opened flower did not affected by SNP treatments as compared to control (p<0.01). Values followed by the same letter within a column indicate they are not significantly different (p< 0.01) by Least Significant Difference (LSD). Table 1 - Effect of different concentrations of sodium nitroprusside (as nitric oxide donor) on vase life, flower opening and floral abscis- sion in cut Gladiolus flowers (Gladiolus grandiflorus cv. White Prosperity) Treatments Vase Life (days) Full-opened flower (%) Un-opened flower (%) Floral abscission (%) Sodium nitroprusside 0 µM 3.0 c 58.60 b 19.21 a 24.39 a Sodium nitroprusside 25µM 3.0 c 60.33 ab 15.51 a 22.99 a Sodium nitroprusside 50 µM 3.33 c 69.83 ab 14.83 a 18.02 a Sodium nitroprusside 75 µM 4.0 bc 66.92 ab 15.87 a 17.19 a Sodium nitroprusside 100 µM 3.33 c 72.38 ab 10.52 a 19.72 a Sodium nitroprusside 125 µM 5.66 ab 74.81 ab 6.38 a 18.79 a Sodium nitroprusside 150 µM 7.33 a 80.25 a 6.52 a 13.21 a Kazemzadeh-Beneh et al. - Postharvest physiology of cut gladiolus flower “White prosperity” 425 significantly increased water uptake (12.33±0.56 and 12.69±1.51 ml day-1 FW) compared to the control (9.16±0.81 ml day-1 FW) resulted in 34.60% and 38.53% increase in vase solution uptake on day 1, respectively; however, they were also conserved the same manner on day 4 or day 8. In contrast, the low concentration did not sufficiently play protective role to inhibit water losses on 4 days, which was also detected that the 25 and 50 μM accelerated water losses, even faster than controls, especially on 8 days. Lipid peroxidation The data, belong to MDA concentration of flowers petals representing the level of lipid peroxidation is revealed in figure 2A. Measurement of MDA demon- strated that vase solutions containing 125 and 150 μM SNP significantly decreased MDA production in comparison to control (p<0.01). Overall, it was pre- dictable that White Prosperity without treatment (control) showed higher MDA concentration than the SNP treatments. Results found inversely correlation between lipid peroxidation and higher SNP concen- tration. At any specified level of SNP concentration, the production of lipid peroxidation product was less- er in treatments at the end experiment (8 days) com- parison to control. Thus, the vase solutions having As shown in figure 1A, RFW (data normalized; 4.6 is equal to 100%) in cut gladiolus flowers of control gradually was declined during the vase life period, while the decline in RFW was no observed by SNP treatments throughout the vase life. It is notable that the RFW of the SNP treatments solutions except to 125 μM at three point time did not significantly dif- ference with those in control solutions at initial point time (day 1). So, the presence of SNP in vase solu- tions displayed a protective role to the inhibition of RFW decline in vase life period, even when the vase l i f e o f c u t f l o w e r s e n d e d . O n l y S N P w i t h concentration 125 μM (5.60±0.08) in vase solution prolonged the RFW 22.55% higher than other con- centrations on day 4 or day 8 in comparison to those in controls at initial time (Fig. 1A). Senescence is a process characterized by water loss and desiccation of plant tissues. During vase life period, water uptake gradually was declined in both some of the SNP treatments (25, 50, and 75 μM) and control cut flowers (Fig. 1B). Generally, the water uptake with SNP concentration 100, 125, 150 μM were higher than those under control condition, respectively (p<0.01). At the initial point time (day 1) of vase life, the White Prosperity showed a rapid response to high SNP concentrations for promoting water uptake; however, the water loss was not observed during its vase life period. The vase solu- tions containing SNP at concentration 125, 150 μM Fig. 1 - The effects of different concentrations of sodium nitro- prusside (as nitric oxide donor) on physiological changes of Gladiolus grandiflorus cv. White Prosperity cut flowers during vase life. Data are means of three replications. Vertical bars indicate standard deviation. Fig. 2 - Effect of different concentrations of sodium nitroprussi- de (as nitric oxide donor) on Malondialdahyde (MDA), as an indicator of lipid peroxidation, total soluble protein in Gladiolus grandiflorus cv. White Prosperity cut flowers during vase life. Vertical bars with the same letters did not show significantly different using LSD method at P<0.01 significant level. 426 Adv. Hort. Sci., 2018 32(3): 421-431 SNP at concentration 125, 150 μM significantly declined the lipid peroxidation of cell membrane (0.463±0.04 and 0.61±0.04 mmol g-1 FW) compared to the control (1.55±0.07 mmol g-1 FW) resulted in 70.13% and 60.65% decline in product induction of lipid peroxidation, MDA, on days 8 prior to senes- cence appearance in cut flowers. Total soluble proteins The chemical analysis for TSP of the flower petals exhibited that SNP significantly increased the TSP dur- ing vase life period in comparison with control (p<0.01) (Fig. 2B). The protein degradation of flower petals in control was higher than SNP treatments; however, the total soluble protein gradually was declined in control across days. Thus, not only SNP lead to help to the inhibition of protein degradation in flowers petals on day 4 or day 8, but also it caused in delaying the senescence of gladiolus flowers. So, all of the SNP treatments except to 150 μM displayed a pro- tective or maintain role for protein degradation in cut flowers. Overall, only increase in TSP was observed with 150 μM SNP and also was recorded highest TSP for its on day 8; however, the protein degradation did not occurred by 150 μM SNP treatment during vase life period. Furthermore, the prolong vase life of White Prosperity flowers can be strongly associated with increasing in TSP and inhibiting from its degrada- tion in flower petals during vase life. Enzymatic antioxidant and non-enzymatic antioxi- dant activities ANOVA analysis with mean comparison showed that antioxidant enzymes and non-enzymatic antioxi- dant activities in flower petals differed significantly between control and treatments in White Prosperity (p<0.01). As expected, the PPO activity (U/mg FW) was continually increased in control during vase life, which this tendency was also approximately found for 25 μM (Fig. 3A). The low concentration from 25 to 75 μM did not sufficiently decrease the PPO activity in flower petals comparison to control (p<0.01). So, the decrease in PPO activity was observed by 100, 125, and 150 μM treatments on day 4 or day 8, respectively; however, increasing SNP concentration in vase solution resulted in markedly decreasing PPO activity in comparison to controls at initial time of vase life (p<0.01). It can be predictable that the posi- tive effect of SNP on maintaining or decreasing PPO activity was high dose-dependent. It is now well rec- ognized that high PPO activity accelerate to senes- cence and to induce browning in plant tissues. Generally, the high concentrations of SNP to White Prosperity cut flowers, check the activity of PPO enzyme, lead to help in delaying the senescence of gladiolus flower via preventing the PPO activity com- pared to control (p<0.01). As shown in figure 3B, the POD activity (U/mg FW) significantly decreased in control flowers throughout vase life, while the CAT activity (U/mg FW) in control flowers displayed a con- stant tendency at all of the 3 point time of vase life (Fig. 3C) comparison to SNP treatments(p<0.01). It is appears that all of the treatments except to 125 μM significantly played a protective role to conserve the decrease of POD activity during vase life (p<0.01). The highest POD activity obtained by 125 μM on 8 days, according to LSD test at p<0.01. However, the positive effect of SNP on POD activity was dose- and time-dependent: 125 μM was observed to be the optimal concentration for POD activity on day 4. Thus, in White Prosperity, low concentrations did not adequately increase POD activity, which was also found for concentrations higher than the optimal lev- els (Fig. 3B). In concerning about CAT activity, the positive relation was detected between CAT activity and SNP treatments; however, increasing in SNP con- centration resulted in increasing CAT activity, espe- cially on day 4 or day 8, compared to control (p<0.01) (Fig. 3C). The results of the present study indicated that with more addition SNP concentration by 100 to 150 μM into vase solution was lead to positively increase in CAT activity at each of three time points during vase life. The results of LSD test (p<0.01) indicated that TMA degradation was gradually happened in control flowers during vase life (Fig. 3D). The SNP treatments not only significantly prevented from the TMA degra- dation but also they were greatly enhanced the TMA p r o d u c t i o n o v e r 8 d a y s , c o m p a r e d t o c o n t r o l (p<0.01). At the during vase life, the low concentra- tions of SNP demonstrated a protective role to inhibit TMA degradation in flower petals in comparison to control (p<0.01). The furthest increase in the TMA production was archived for 125 μM (0.273 ± 0.037 mg l-1), and 150 μM (0.193±0.015 mg l-1) with a signif- icant difference compared to control, respectively. Hence, improved TMA in flower petals likely to POD activity was dose- and time-dependent: 125 μM was observed to be the optimal concentration for TMA content. Thus, in White Prosperity, low concentra- tions did not adequately increase TMA content, which was also detected for concentrations higher than the optimal levels on day 4 or day 8. 427 Kazemzadeh-Beneh et al. - Postharvest physiology of cut gladiolus flower “White prosperity” Pearson correlation analysis reveals interactions between physiological, biochemical and antioxidant system related traits In order to arrange for an overview of the associa- tions between physiological, biochemical traits, and antioxidant system activity, the Pearson correlation test used for analyzing and thus was investigated all of the significant associations, as presented in Table 2. From this analysis 23 positive and 11 negative sig- nificant correlations was achieved. Among them, some correlations were expected, such as the posi- tive and negative correlations observed between antioxidant system activity, for example, CAT activity and TMA content (r= 0.89, p<0.01), and PPO activity and TMA content (r= -0.95, p<0.01) on days 8, respectively. With regard to physiological traits, the results of paired linear correlation indicated that RFW was positively correlated with CAT (r= 0.85, p<0.05 on days 4), and POD activity (r= 0.86, p<0.05 on days 4 and r= 0.81, p<0.05 on days 8), and TMA content (r= 0.79, p<0.05 on days 8), while was nega- tively correlated with PPO activity on day 4 (r= -0.80, p<0.05) and day 8 (r= -0.87, p<0.05) of the White Prosperity vase life. Also, the Pearson correlation of water uptake with CAT activity (r= 0.80, p<0.05 on days 1; r= 0.82, p<0.05 on days 4; r= 0.86, p<0.05 on days 8) and with TMA content (r= 0.81, p<0.05 on days 8) was positive significant, whereas displayed a negative significant with PPO activity (r= -0.79, p<0.05 on days 4 and r= -0.85, p<0.05 on days 8) (Table 2). However, suggesting that the SNP treat- ment is a key inhibitor to water loss and an inducer to antioxidant system for delaying the senescence of gladiolus flowers during vase life, especially on 4 and days 8. Total soluble protein had a positive correlation with TMA content and a negative correlation with PPO activity. TMA indicated a positive correlation with water uptake, RFW, total soluble protein, and CAT activity and a negative correlation with PPO and POD activity. CAT activity was positively correlated with physiological traits, TMA and negatively corre- lated with PPO and POD activity. However, according to the results of Pearson correlation, suggesting that SNP might be an important protective or inducer involved in the physiological, biochemical process and antioxidant system in White Prosperity vase life that can be alleviate to water loss, RFW, and to browning process, which lead to early senescence appearance. 4. Discussion and Conclusions The postharvest longevity of cut flower has a criti- cal importance in determining the value of crop. Recently, SNP, a NO donor known to be a signal mol- ecule involved in biotic and abiotic stress tolerance, has been increasingly used to extend the vase life of Fig. 3 - Evaluating the effects of different concentrations of sodium nitroprusside (as nitric oxide donor) on enzyma- tic and non-enzymatic antioxidant system changes during vase life period of Gladiolus grandiflorus cut flowers. Vertical bars indicate standard deviation. 428 Adv. Hort. Sci., 2018 32(3): 421-431 cut flowers, such as rose, gladiolus, and carnation (Naing et al., 2017). First data concerning about application exogenous SNP to improve vase life of G. grandiflora cv. Snow Princess cut flower has been reported by Dwivedi et al. (2016). It is generally accepted that different genotypes or cultivars might indicate different physiological or biochemical responses to exogenous SNP, which is the effects induced by it may be rely on dose- and cultivar- dependent. Some published evidences supports NO acting as a negative regulator during leaf senescence, but also there is opposite result in this regard; NO enhances flower abscission and senescence in cut racemes of Lupinus havardii Wats (Sankhla et al., 2003; Guo and Crawford, 2005). Thus, the properly effects of SNP on enhancing physiological and bio- chemical processes for one cultivar, may not be suit- able for another, which is due to differences in their genetic background. This aspect has also been con- firmed by Naing et al. (2017), who found that SNP dose that was best for one cultivar of Gerbera cut flower was not suitable for another; thus, variation in the optimal dose of SNP among cultivars for the enhancement of their vase life could result from dif- f e r e n c e s i n t h e i r g e n e t i c b a c k g r o u n d . H e n c e , whether SNP participate in improving of cut flowers of White Prosperity cultivar has not been yet recon- noitered. Therefore, in the current study, we investigated the role of SNP in the enhancement of physiological, biochemical responses, and antioxidant activity to extend vase life of Gladiolus grandiflorus cv. White Prosperity cut flower. Cut flower senescence is linked to a sequence of highly regulated physiological and biochemical processes such as degradation of pro- teins, DNA content, peroxidation lipids and mem- brane leakage, degradation of macromolecules, cellu- lar decompartmentalization, floral abscission, color change, leaf yellowing, and weight loss (Buchanan- Wollaston et al., 2003; Nasibi et al., 2014). In this study, results of our findings revealed that the physi- o l o gi c a l , b i o c h em i c a l , a n d a n t i o x i d a n t a c t i vi t y induced by SNP in White Prosperity cultivar were more different than those induced in Snow Princess cultivar, a previous study by Dwivedi et al. (2016), which it may be due to differences in their genetic background. Hence, in present study, SNP was signifi- cantly promoted the vase life of ‘White Prosperity’ cut flowers through help to delay the senescence appearance and desiccation on tissue or organ level; however, it effects were discovered to be dose- and time-dependent. Vase life positively associated with Table 2 - Pearson correlation between physiological and biochemical characteristics of Gladiolus grandiflorus cv. White Prosperity cut flowers affected by sodium nitroprusside during vase life period WU= water uptake; RFW= relative fresh weight; TSP= total soluble protein; PPO= polyphenol oxidase activity; POD= peroxidase activity; CAT= catalase activity; TMA= total monomeric anthocyanin, the 1, 2, and 3 representing vase life time for each variable on day 1, day 4, and day 8. NS, *, ** non-significant, correlation is significant at the 0.05 and the 0.01level, respectively. RFW1 has no computed because at least one of the variables was constant. Traits WU1 WU2 WU3 RFW2 RFW3 TSP1 TSP2 TSP3 PPO1 PPO2 PPO3 POD1 POD2 POD3 CAT1 CAT2 CAT3 TMA1 TMA2 TMA3 WU1 1 0.932 ** 0.919 ** 0.499 NS 0.595 NS -0.499 NS 0.261 NS 0.408 NS 0.288 NS -0.584 NS -0.713 NS -0.741 NS 0.152 NS 0.152 NS 0.803 * 0.746 NS 0.927 ** 0.421 NS 0.655 NS 0.732 NS WU2 1 0.849 * 0.698 NS 0.753 NS -0.383 NS 0.517 NS 0.616 NS 0.48 NS -0.579 NS -0.801* -0.666 NS 0.347 NS 0.347 NS 0.752 NS 0.829 * 0.981 ** 0.434 NS 0.752 NS 0.854 * WU3 1 0.627 NS 0.722 NS -0.388 NS 0.430 NS 0.553 NS 0.40 NS -0.791* -0.850* -0.787* 0.271 NS 0.271 NS 0.780 * 0.871 * 0.862 * 0.25 NS 0.697 NS 0.811 * RFW2 1 0.988 ** 0.208 NS 0.747 NS 0.687 NS 0.802 * -0.575 NS -0.809* -0.295 NS 0.866 * 0.866 * 0.296 NS 0.854 * 0.656 NS 0.233 NS 0.534 NS 0.745 NS RFW3 1 0.100 NS 0.713 NS 0.677 NS 0.774 * -0.663 NS -0.872* -0.406 0.817 * 0.817 * 0.352 NS 0.892 ** 0.724 NS 0.311 NS 0.565 NS 0.794 * TSP1 1 -0.009 NS -0.208 NS 0.071 NS 0.36 NS 0.342 NS 0.615 NS 0.585 NS 0.585 NS -0.514 NS -0.215 NS -0.436 NS -0.333 NS -0.319 NS -0.405 NS TSP2 1 0.971 ** 0.558 NS -0.64 NS -0.75 NS -0.432 NS 0.445 NS 0.445 NS 0.405 NS 0.685 NS 0.543 NS -0.062 NS 0.771 * 0.820 * TSP3 1 0.526 NS -0.693 NS -0799* -0.561 NS 0.299 NS 0.299 NS 0.591 NS 0.751 NS 0.642 NS -0.073 NS 0.851 * 0.889 ** PPO1 1 -0.272 NS -0.615 NS 0.056 NS 0.747 NS 0.747 NS 0.16 NS 0.775 * 0.365 NS 0.068 NS 0.154 NS 0.507 NS PPO2 1 0.897** 0.868* -0.21 NS -0.21 NS -0.486 NS -0.7 NS -0.682 NS -0.286 NS -0.734 NS -0.847* PPO3 1 0.739 NS -0.45 NS -0.45 NS -0.556 NS -0.910** -0.832* -0.36 NS -0.742 NS -0.956** POD1 1 0.152 NS 0.152 NS -0.688 NS -0.532 NS -0.786* -0.363 NS -0.817* -0.791* POD2 1 1.000** -0.14 NS 0.548 NS 0.269 NS 0.185 NS 0.086 NS 0.318 NS POD3 1 -0.14 NS 0.548 NS 0.269 NS 0.185 NS 0.086 NS 0.318 NS CAT1 1 0.661 NS 0.749 NS -0.124 NS 0.779 * 0.682 NS CAT2 1 0.786* 0.125 NS 0.646 NS 0.862 * CAT3 1 0.482 NS 0.823 * 0.894 ** TMA1 1 0.084 NS 0.3 NS TMA2 1 0.887 ** TMA3 1 Kazemzadeh-Beneh et al. - Postharvest physiology of cut gladiolus flower “White prosperity” 429 RFW, water uptake, TSP content, TMA, enzyme antioxidant activity and lipid peroxidation. At the start of vase life, there was a noticeably increase and then a constant tendency in water uptake of White Prosperity cut flowers during their vase life, which suggested that SNP might have a protective role in cut flowers against water losses stress (Fig. 1B). The rapid increase in initial water uptake was dose-and time-dependent, while increase in RFW was more dose-dependent (Fig. 1A). The 125 μM concentration was observed to be the optimal concentration for increasing in RFW. The RFW increase obtained in White Prosperity is in isagreement with results pro- nounced by Dwivedi et al. (2016) in Snow Princess. The inhibition or improvement in RFW across days under NO condition is probbly attributed to the excessive potential of water uptake, leading to a sta- bility or promote in cell turgidity pressure, which restricts burning from reserved carbohydrates in res- piration and limits fresh weight reduction. An associ- ation of improved water uptake and inhibited fresh weight reduction has been reported by Vajari and Nalousi (2013) in carnation and Naing et al. (2017) in gerbera cut flower. Overall, the ‘White Prosperity’ in SNP (150 μM) had prolonged vase life over control, which was strongly associated with increased water uptake and improved RFW. The damage to the plant cell’s biomembrane liable to senescence process, decrease in the ratio of unsaturated fatty acids, change mobility of the cell membrane, and generate free radicals are resulted in an increase in the concentration of Malondialdahyde (MDA), which is an indicator of lipid peroxidation and of injury to the plant cell membrane (Chen, 2009). So, the higher membrane stability plays a key role in inhibiting leakage of electrolytes, sugars, pigment, solute leakage, and also lipid peroxidation as well as in delay senescence during gladiolus cut flowers postharvest (Ezhilmathi et al., 2007; Ghadakchiasl et al., 2017). Our results showed that the change in membrane stability and lipid peroxidation occurrence resulting from the MDA production were alleviated by SNP concentrations (150, 125 μM) on day 7 after treatment and therefore protected and reduced White Prosperity MDA production in cell membrane (Fig. 2A). These results are in agreement with those reported earlier by Mansouri (2012), who suggested that SNP prolonged the vase life of chrysanthemum flowers, which was accompanied by decreasing in the electrolyte leakage, levels of MDA and lipid peroxida- tion. Indeed, the role of NO in prevention of lipid per- oxidation is related to the ability of NO to react with lipid alcoxyl (LO•) and lipid peroxyl (LOO•) radicals and stop the chain of peroxidation in a direct fashion (Beligni and Lamatina, 1999). The role of SNP in reducing membrane lipid peroxidation has previously been stated by Liao et al. (2012) and Dwivedi et al. (2016). Many researchers have been shown that protein degradation and also shortage protein due to con- sumption it instead of soluble carbohydrate during senescence process for respiration in petals are the most important causes for shortening cut flowers vase life (Rezvanypour and Osfoori, 2011). In addi- tion, SNP significantly maintained the TSP degrada- tion in cut flowers petals, while in absence SNP increased the TSP degradation to a greater rate than SNP treatments on day 4 and day 8 (Fig. 2B). The TSP measured in vase solution supplemented by 150 μM was distinctly higher than those placed in controls on day 8. The proteins are the basic components of all cell activities, their reduction degrades enzymes and causes higher production of free radicals, as well as reducing protein synthesis (Saed-Moucheshi et al., 2014). Therefore, it was clear that the proteins degradation during vase life significantly inhibited by SNP supplements in the vase life of ‘White Prosperity’. The increase or protect of TSP degradation by SNP application in strawberry (Ghadachiasl et al., 2017) and in peanuts (Verma et al., 2010) has also been claimed. Earlier studies have been confirmed that SNP may either be directly scavenging ROS and thus decreas- ing lipid peroxidation, or it may be modulating the activity of antioxidant system (Beligni and Lamatina, 1999; Saed-Moucheshi et al., 2014). Various studies have demonstrated that the vase life of cut flowers is modulated by antioxidant enzymes and non- enzy- matic antioxidant activities (Vajari and Nalousi, 2013). Thus, supplemented vase solutions with SNP stimulated a higher enzymatic or non-enzymatic antioxidant activity in flower petals during vase life period. The PPO catalyzes the browning reaction and results in the formation of quinine, which is subse- quently polymerized to varying degree leading to production of brown pigments (Dubravina et al., 2005). The PPO activity greatly was reduced by SNP, while the POD and CAT activity greatly promoted by SNP during progress senescence (Fig. 3). The results were in accordance with the findings of Ghadakchiasl et al. (2017) and Dwivedi et al. (2016). Indeed, the NO synthesized by SNP in tissue plant acts signaling Adv. Hort. Sci., 2018 32(3): 421-431 430 molecule to enhance the enzymatic antioxidant activ- ity such as SOD and CAT and ultimately protects pro- teins degradation as well as lipid peroxidation against free radicals. So, POD and CAT high activity induced by SNP showed a negatively correlation with PPO activity and thus they blocked PPO activity during vase life (Table 2). Approximately, high and markedly negative between PPO and more traits examined in current study were also detected in Pearson correla- tion analysis. Furthermore, TMA degradation in flower petals protected by SNP, while TMA degrada- tion increasable induced in flower petals without presence SNP during vase life period. However, posi- tive effects induced by SNP in both POD activity and TMA were dose- and time-dependent, therefore, the 125 μM was selected as an optimal concentration for TMA and POD activity (Fig. 3B, D). Antioxidant com- pounds such as vitamin C, glutathione, and antho- cyanin plays vital role, as non-enzymatic system, in protecting cell against destructive chemical com- pounds such as free radicals and reactive oxygen species (ROS) that are constantly produced by the cell metabolism and their concentration increases under stress conditions (Kazemzadeh et al., 2015). However, SNP increased TAM content at any time and level of SNP concentration in comparison to con- trols. The high and significantly positive correlation between TMA anthocyanin with CAT activity and total soluble protein was obtained by pearson analy- sis (Table 2). With progress senescence during vase life, TMA probably scavenged free radicals due to oxidative stress, consequently, inhibited more deteri- oration of membrane and protein degradation in flower petals. In conclusion, vase life period in the G. grandi- florus cv. White Prosperity cut flower is likely to be associated with many parameters, particularly fresh weight content, water uptake, enzymatic or non- enzymatic antioxidant activities, membrane stability and lipid peroxidation. Furthermore, it was found that positive effects induced by SNP on vase life dis- tinctly were dose- and time-dependent and were also genetic back ground cultivar-dependent in compara- tive responses between White Prosperity with Snow Princess, which has previously been reported by Dwivedi et al. (2016). 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