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Exogenous sodium nitroprusside treatment of broccoli florets extends shelf life, enhances antioxidant enzyme activity, and inhibits chlorophyll-degradation
Postharvest Biology and Technology 116 (2016) 98–104
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Exogenous sodium nitroprusside treatment of broccoli florets extends shelf life, enhances antioxidant enzyme activity, and inhibits chlorophyll-degradation Junyan Shia,b , Lipu Gaoa,b , Jinhua Zuoa,b , Qing Wanga,b,* , Qian Wanga,b , Linlin Fana,b a
National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing Key Laboratory of Fruits and Vegetable Storage and Processing, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 100097, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 June 2015 Received in revised form 15 January 2016 Accepted 18 January 2016 Available online 8 February 2016
The effect of an exogenous application of sodium nitroprusside (SNP) on the shelf life, antioxidant enzyme and chlorophyll-degrading enzyme activity, and chlorophyll-degradation related gene expression was investigated in stored broccoli. The preliminary results indicated that the 200 mmol L1 SNP treatment had the greatest effect on extending shelf life of broccoli florets so this concentration was used in the remainder of the study. The SNP treatment delayed chlorophyll degradation, thus color was maintained and shelf-life was extended. The activity of the antioxidant enzymes, catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) was all enhanced while glutathione reductase (GR) activity was inhibited throughout the storage. Relative to the untreated control, the SNP treatment suppressed the activity of the chlorophyll-degrading enzymes, chlorophyllase (Chlase), chlorophylldegrading peroxidase (Chl-POX), Mg-dechelatase (MD), and pheophytinase (PPH), and also suppressed chlorophyllase I (BoCHL1), chlorophyllase II (BoCHL2), chlorophyllase III (BoCLH3) and pheophorbide a oxygenase (BoPAO) gene expression during the entire storage period. In summary, 200 mmol L1 SNP treatment of broccoli extends shelf life, enhances oxidative stress tolerance by enhancing the activity of antioxidant enzymes, and inhibits the activity of chlorophyll-degrading enzymes and related gene expression. The combined effect delayed the yellowing of broccoli florets by inhibiting chlorophyll degradation, thus extending the shelf life of broccoli florets. ã 2016 Published by Elsevier B.V.
Keywords: Sodium nitroprusside Broccoli Antioxidant enzyme activity Chlorophyll-degrading enzyme activity Gene expression
1. Introduction Broccoli (Brassica oleracea var. Italica), a cruciferous vegetable, contains a high level of antioxidant compounds. It is also rich in vitamins, and anti-carcinogenic compounds, such as glucosinolates, and sulforaphane nitrile (Williams et al., 2008). However, it is a highly perishable product, and florets undergo rapid yellowing at room temperature after harvest (Hansen et al., 2001), thus reducing the shelf life of the florets and resulting in a low-quality product (Funamoto et al., 2006). The yellowing of broccoli florets, the symptom of postharvest senescence, is accompanied by a loss in fresh weight, chlorophyll content, sugars, and proteins (Costa
* Corresponding author at: National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China. E-mail address: [email protected] (Q. Wang). http://dx.doi.org/10.1016/j.postharvbio.2016.01.007 0925-5214/ ã 2016 Published by Elsevier B.V.
et al., 2005). There is also a loss in aroma, a decrease in amino acid content (Hansen et al., 2001), a decrease in total aliphatic and indole glucosinolates, and a general reduction in antioxidant compounds (Jia et al., 2009). Additionally, lipid peroxidation and protein degradation increased (Page et al., 2001). Oxidative processes play an important role in plant senescence, thus antioxidant enzymes, such as catalases (CAT), superoxide dismutases (SOD), peroxidases (POD), and the ascorbate-glutathione, have evolved in plants to combat oxidative stress and delay senescence (Lemoine et al., 2010). Yellowing of broccoli florets results from a loss and degradation of chlorophyll (Chl). Chl a is degraded by enzymes that include chlorophyllase (Chlase) (Harpaz-Saad et al., 2007), chlorophylldegrading peroxidase (Yamauchi et al., 2004), Mg-dechelatase (MD) (Suzuki et al., 2005), or another Mg-dechelating substance (MDS), pheophorbide (pheide) a oxygenase (PaO), and red Chl
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catabolite reductase (Hörtensteiner, 2006). Chl degradation occurs mainly within the chloroplast. Moreover, the peroxidase-mediated Chl degradation may also occur in the vacuole (Yamauchi et al., 2004; Hörtensteiner, 2006). Several approaches have been used to delay the senescence of broccoli florets, including heat treatment (Lemoine et al., 2010), modified atmosphere packaging (Jia et al., 2009), UV irradiation (Costa et al., 2006; Aiamla-or et al., 2009, 2012), and chemical treatments such as ozone and 1-methylcyclopropene (1-MCP) (Forney et al., 2003). Nitric oxide (NO) is an important signaling molecule with diverse physiological functions for plants, participating in various cellular processes, such as respiratory metabolism, growth and development, as well as maturation and senescence (Lamattina et al., 2003). Some researchers found that NO gas or sodium nitroprusside (SNP, as a donor of exogenous NO) treatment, delayed ripening, senescence and enhanced the resistance in horticulture crops (Wills et al., 2008; Wu et al., 2014; Iakimova and Woltering, 2015). For instance, Leshem et al. (1998) originally reported that NO-treated broccoli markedly retained green color and firmness compared to the air control and the ethylene-treated broccoli. The senescence-delaying effects of NO were also observed in carnation, Geraldton wax flowers (Chamelaucium uncinatum Schauer) and waratah flowers (Telopea speciosissima, Proteaceae) (Zeng et al., 2011; Leshem et al., 1998). However, the metabolic and physiological responses of broccoli to NO treatment remains still poorly understood. The overall objective of present study was to determine the effect of NO treatment (via the exogenous application of SNP) on broccoli florets stored at 20 C. More specifically, the effects of SNP on broccoli floret quality parameters, antioxidant enzyme and chlorophyll-degrading enzyme activity, as well as chlorophylldegradation related gene expressions during storage were investigated. 2. Materials and methods 2.1. Plant material and treatments Broccoli (B. oleracea var. Italica, ‘Bao-shi’) was harvested from an organic vegetable farm in the town of Xiaotangshan, located in the Shunyi District of Beijing, China, top-iced and directly brought to the laboratory within 3 h. The selected broccoli ranged in diameter from 15 to 20 cm, were uniform in color, had no evidence of insect or disease injury, no mechanically-induced wounds, and all had tight-head clusters. The selected broccoli heads were randomly placed into five groups. One group was treated with distilled water (control) and four groups received exogenous application of 100, 200, 400, and 800 mmolL1 sodium nitroprusside (SNP). Broccoli heads were air dried and then placed in polyethylene film bags (0.03 mm in thickness), with the top folded over, placed in plastic crates, and stored in the dark at 20 1 C and 90% RH. An assessment of shelf life, and a visual color scale rating were made daily in replicates of nine broccoli heads per treatment. The florets, together with about 1 cm of stalk, were cut from the broccoli heads each day and served as a sampling unit. The samples were immediately frozen in liquid nitrogen and stored at 80 C. The frozen florets were used to determine chlorophyll content, antioxidant enzyme activity, chlorophyll-degrading enzyme activity, and the relative expression of genes coding for proteins involved in chlorophyll degradation. 2.2. Determination of shelf life The shelf life of broccoli florets was determined according to the previous study (Yuan et al., 2010). A percentage of 30% yellowing florets was designated as the end of shelf life.
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2.3. Measurement of visual color rating scale The color of broccoli florets was measured by a visual assessment of changes from green to yellow as previously described (Rangkadilok et al., 2002). A color rating scale from 0 to 9 was adopted to indicate the general percentage of yellowing in the broccoli head, where 0 indicates all dark green, 1 indicates 3– 5% yellowing, 5 indicates 50% yellowing of the head, 7 indicates 75% yellowing of the head, and 9 indicates 100% yellowing of the broccoli heads. 2.4. Total chlorophyll content Frozen floret tissues were homogenized in acetone:ethanol (2:1) using a mortar and pestle. The homogenate was then filtrated and the filtrate was used to measure chlorophyll content according to Sun et al. (2012). The optical density at 645 nm and 643 mm were recorded for each sample. 2.5. Antioxidant enzyme activity Three grams of frozen flower tissue was extracted with 15 mL 0.1 molL1 phosphate buffer (PBS, pH 7.8, containing 0.5% polyvinylpyrrolidone), then centrifuged at 12,000 g for 20 min at 4 C. The supernatant was collected and used for enzyme assay. Catalase (CAT) activity was measured by determining the decrease in absorbance at 240 nm due to H2O2 consumption as described by Azevedo et al. (2007). The reaction mixture contained: 1.0 mL of 0.3% H2O2, 1.9 mL of 0.05 molL1 phosphate buffer (PBS, pH 7.8), and 0.1 mL of sample. Peroxidase (POD) activity was measured according to Bai et al. (2006). One gram of frozen floret tissue was homogenized in 8.0 mL of 0.05 M PBS (pH 7.8). The reaction mixture contained: 1.0 mL of 0.3% H2O2, 1.0 mL of 0.05 M PBS (pH 7.8), 0.9 mL of 0.2% guaiacol, and 0.1 mL of sample. The increase in absorbance at 470 nm over one minute was determined. Glutathione reductase (GR) activity was assayed by measuring NADPH oxidation in the presence of oxidized glutathione (Ognjanovi c et al., 2010). The reaction mixture consisted of 2.7 mL PBS (pH 7.5, containing 1 mmol L1 EDTA), 0.1 mL of 5 mmol L1 oxidized glutathione, 0.2 mL of sample, and 40 mL of 4.0 mmol L1 NADPH. GR activity was determined as a change in absorbance at 340 nm over 1 min. Ascorbate peroxidase (APX) activity was determined by measuring the decrease in absorbance of ascorbic acid (AsA) at 290 nm (Zhang et al., 2010). The reaction mixture contained 2.6 mL of PBS (pH 7.5, containing 0.1 mmol L1 EDTA and 0.5 mmol L1 AsA), 0.1 mL of sample, and 0.3 mL of 2 mmol L1 H2O2. 2.6. Chlorophyll-degrading enzyme activity An acetone-derived powder of floral tissues was prepared by the method of Aiamla-or et al. (2012). About 1 g broccoli floral tissues was homogenized in cold acetone (20 C). The homogenate was placed at 20 C for 15 min, then centrifuged at 12,000 g for 5 min at 4 C. The precipitate was then washed twice with cold acetone. The resulting precipitate was used as the source of acetone powder in the following assays. Chl a was bought from sigma company, and then 200 mg mL1 Chl a was prepared in acetone. Chlorophyllin a (Chlin) was prepared with 30% KOH in methanol from chl a in petroleum ether. Pheophytin (Phy) a was prepared by the acidic reaction from Chl a, using 0.1 M hydrochloric acid. The Phy a concentration was measured spectrophotometrically at 409 nm (Aiamla-or et al., 2012). Chlorophyll-degrading enzymes were extracted and measured as described by Aiamla-or et al. (2010).
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Chlorophyllase (Chlase) activity was determined as described by Aiamla-or et al. (2010). The reaction mixture contained 0.5 mL PBS (pH 7.5), 0.2 mL of 200 mg mL1 Chl a acetone solution, and 0.5 mL of enzyme extract. The reaction mixture was incubated in a water bath at 25 C for 40 min. The enzyme reaction was then stopped by adding 4 mL of acetone to the reaction mixture. Chlorophyllide (Chlide) was separated from the reaction mixture by adding 4 mL of hexane. The lower phase contained Chlide a. The level of Chlide was determined by measuring absorbance at 667 nm per unit. Chlorophyll-degrading peroxidase (Chl-POX) activity was determined as described by Asumi et al. (2010). The reaction mixture contained 0.5 mL of enzyme solution, 0.2 mL of 80 mg mL1 Chl a acetone solution, 0.1 mL of 0.3% H2O2, 0.1 mL of 1.0% Triton-X 100, 0.1 mL of 800 mmolL p-coumaric acid, and 1.5 mL of 0.1 molL1 PBS (pH 5.5). Activity was determined by measuring the decrease of Chl a by measuring the change in absorbance at 668 nm per unit at 25 C. Mg-dechelatase (MD) activity was measured according to Suzuki and Shioi (2002), using Chlin. Pheophorbin a formation was determined by measuring absorbance at 686 nm (Costa et al., 2005). The reaction mixture, containing 0.75 mL of 50 mmol L1 Tris–HCl buffer (pH 8.0), 0.3 mL of Chlin, and 0.2 mL of enzyme solution, was incubated at 37 C for 10 min. MD activity was expressed as the increment change in OD at 686 nm per minute under the test conditions. Pheophytinase (PPH) activity was measured based on the methods described by Schelbert et al. (2009) and Aiamla-or et al. (2012). The reaction mixture contained 0.35 mL of enzyme solution, 75 mL of 2.0% Triton-X 100, 0.1 mL of Phy a (10 mmol L1), and 0.70 mL of 50 mmol L1 Tris–HCl (pH 8.0). The reaction was incubated in darkness for 90 min at 25 C. The reaction was stopped by addition of acetone to the reaction mixture. Pheophorbide (pheide) a was separated from Phy a by the addition of hexane to the reaction mixture. Pheide a concentration in the acetone layer was measured spectrophotometrically at 667 nm. 2.7. RNA extraction and RT-qPCR analysis The frozen broccoli frozen florets tissues were first ground into a powder in liquid nitrogen. Total RNA was extracted from the powdered tissue using Trizol reagent (Invitrogen) and used as a template for cDNA synthesis utilizing SuperScript1 RT (Invitrogen). The resulting cDNA was stored at 80 C and subsequently used as a template for PCR using Ex Taq1 polymerase (Invitrogen) and gene-specific primers. The relative expression of the following genes was analyzed by RT-qPCR: Actin (BoAct, Genbank No. AF044573); chlorophyllase I (BoCLH1, GenBank NO. AF337544), chlorophyllase II (BoCLH2, GenBank NO. AF337545; chlorophyllase III (BoCLH3, GenBank NO. AF337546); and a Phride a oxygenase (BoPAO, GenBank NO. AF337544). The cDNA was used as a template for RT-qPCR utilizing UltraSYBR Mixture (Kangwei) in a Roche LightCycler1 480 machine according to the manufacturer’s instructions. Gene-specific primes were designed using Primer Express 5.0 (Applied Biosystem). Actin was used as a reference gene for standardization of all the results Aiamla-or et al. (2012). The gene-specific primers used to amplify each gene were as follows: BoAct, forward 50 CTTGCAC CAAGCAGCATGAA-30 , reverse 50 -AGAATGGAACC ACCGATCCA-30 ; BoCLH1 forward 50 -CCCCGTCGTCTTATTCTT-3, reverse 50 -TGTCCCTTCGTAGCCTTA-30 ; BoCLH2 forward 50 -CCACAAGT AACACCCAACC-30 , reverse 50 -ACCTTTCCCTGTCCCATC-30 ; BoCLH3 forward 50 -CGGTGG AGGAAGGAGAATA-30 , reverse 50 TTGTGGTGGAAGAAA GTGG-30 ; and BoPAO forward 50 -TCGGAACGATCAGAAATG-30 , reverse 50 -AAGTAGCAGC- CTGTGGAA-30 . Each measurement was performed in triplicate.
2.8. Statistical analysis All statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data from each analysis was subjected to a one-way ANOVA and means separations were compared by LSD test at a significance level of P < 0.05. No significant interactions between the treatments and time of sampling were identified. Data are presented as means and standard deviations. 3. Results 3.1. Effect of SNP treatment on shelf life In this study, 800 mmolL1 SNP treatment significantly decreased the shelf life of broccoli florets, while the other concentrations of SNP extended shelf life (Fig. 1). The greatest increase in shelf life was observed in broccoli florets treated with 200 mmolL1 SNP, which extended the shelf life by one day relative to the untreated control. Based on these results, 200 mmol L1 SNP was selected for more detailed studies. 3.2. Effect of 200 mmolL1 SNP on yellowing The yellowing of broccoli, stored at 20 C rapidly increased over a four-day period (Fig. 2). The SNP treatment, however, significantly delayed the yellowing of broccoli florets. The delay of yellowing by the SNP treatment was clearly observed after two days of storage. 3.3. Chlorophyll content A significant decrease in total chlorophyll content was observed in broccoli florets during storage at 20 C in both groups (control and SNP) (Fig. 3). The level of decrease in chlorophyll content, however, was lower in SNP-treated broccoli florets relative to the control. This difference was evident by day 4 where the total chlorophyll content of the SNP-treated broccoli had decreased by 52.55% from the initial chlorophyll content, while the control exhibited a 74.95% decrease relative to the initial chlorophyll content. 3.4. Antioxidant enzyme activity A decrease in CAT activity of broccoli was observed during the first 2 d of storage at 20 C in the control and SNP-treated groups. This was followed by and increase during days 3 and 4 in both groups (Fig. 4A). CAT activity, however was significantly higher in
Fig. 1. Effect of different concentrations of sodium nitroprusside (SNP) on the shelf life of broccoli florets stored at 20 C. Each bar is the mean sd of three replicate samples, where each replicate consists of nine broccoli heads. Values not sharing a common letter are significantly different at p < 0.05.
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Fig. 2. Percent change in the color (yellowing) of broccoli florets in broccoli heads treated with water (control) or 200 mmolL1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
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(Fig. 4B). POD activity in SNP-treated samples was higher, relative to the control group, especially on days 3 and 4. Levels of POD activity were significantly higher on days 3 and 4 in the SNPtreated group than in the control group. A gradual decrease in GR activity was observed in the broccoli floret tissues of both SNP-treated and untreated, control samples during the 4 d storage period at 20 C (Fig. 4C). SNP treatment of broccoli, however, suppressed the decline in GR activity, except in the 1 d samples. A linear decline in APX activity was observed in both groups of broccoli florets (Fig. 4D), represented by a 46.85% and 21.42% decline, relative to the initial activity, in the control and SNP groups, respectively, over the 4 d period of storage at 20 C. At 4 d, APX activity greatly increased in SNP-treated samples while activity continued to decline in the control. 3.5. Chlorophyll-degrading enzyme activity
Fig. 3. Change in total chlorophyll content in broccoli florets in broccoli heads which had been treated with water (control) or 200 mmolL1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
the SNP group on day 3. No significant differences were observed on day 4. POD activity steadily increased in both the control and SNPtreated samples over the four day period of storage at 20 C
Chlase activity in florets of both the control and SNP-treated groups exhibited a significant decrease after one day at 20 C (Fig. 5A). At 2 d onward, however, the activity was lower in the SNPtreated group than in the control group. Chlase activity increased to 151.03 nmolg1h1 at day 2, from 140.91 nmolg1h1 on day 1, in the control samples, while the activity in SNP-treated florets continued to decrease. Chl-POX activity increased in both the control and the SNP treatment by day 2. After that, however, activity sharply increased in the untreated (control) broccoli but much less in the SNP-treated broccoli. Chl-POX activity remained high in the control group on days 3 and 4 but sharply decreased (day 3) and then increased (day 4) in the SNP-treated group. The level of activity in the SNP-treated groups was significantly lower than in the control group on day 4 (Fig. 5B). MD activity was examined by using Chlin as a substrate. MD activity in the two groups increased during the whole period of storage but increased to a greater extent in the untreated, control group of broccoli, relative to the SNP-treated samples of broccoli (Fig. 5C). The SNP treatment suppressed MD activity in broccoli
Fig. 4. Changes in CAT (A), POD (B), GR (C), and APX (D) activity in broccoli florets of broccoli heads treated with water (control) or 200 mmolL1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
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Fig. 5. Changes in Chlase (A), Chl-POX (B), MD (C), and PPH (D) activity in broccoli florets. Broccoli heads were treated with water (Control) or 200 mmol L1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
florets and delayed yellowing. Initial PPH activity at the time of harvest was approximately 3.86 nmolg1h1, but increased to 4.31 nmolg1h1 and 3.43 nmolg1h1 in control and SNPtreated samples, respectively, after 1 d (Fig. 5D). PPH activity in broccoli florets increased gradually in both groups but was significantly higher in the control group, except on day 4. 3.6. RT-qPCR analysis The relative expression of BoCLH1 gene in the control broccoli florets greatly increased after one day in storage, and then greatly
decreased (Fig. 6A). In contrast, BoCLH1 expression in the SNPtreated group increased only a small amount after 1 d in storage, then decreased during days 2 and 3, and finally increased slightly on day 4. Expression of BoCLH2 gene initially decreased on day 1 and then gradually increased during the remainder of the storage period (Fig. 6B). BoCLH2 expression in the SNP-treated group also exhibited a decrease after 1 day of storage, followed by a slight increase on day 2, a very sharp decrease on day 3, and then a significant increase on day 4, at which time the level of activity was similar in both groups.
Fig. 6. Changes in BoCLH1 (A), BoCLH2 (B), BoCLH3 (C), and BoPAO (D) relative gene expression in broccoli florets. Broccoli heads were treated with water (Control) or 200 mmol L1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
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The expression of BoCLH3 was relatively low in both groups during the first three days of storage but then sharply increased about 6-fold in the SNP-treated group while remaining low in the control group (Fig. 6C). The pattern of expression of BoPAO in the control and SNP-treated broccoli florets was similar over the storage period, except on day 3, at which time expression decreased in SNP-treated samples but remained the same in the control group. Levels of BoPAO expression on day 4 were similar in both the SNP-treated and untreated control samples (Fig. 6D). 4. Discussion Evidences for the signaling roles of NO in plants have been obtained by measuring endogenous NO content and physiological changes after treatment with NO or SNP (Lin et al., 2013; Siddiqui et al., 2011). It can inhibit ethylene biosynthesis, delay ripening and senescence and enhance disease resistance in horticultural plants, including broccoli (Leshem et al., 1998; Soegiarto and Wills, 2004; Eum et al., 2009). In the present study, treatment with 200 mmol L1 SNP was shown to be effective in delaying the senescence. Compared with untreated broccoli, the shelf life prolonged one day (Fig. 1), the yellowing were retarded (Fig. 2) stored at 20 C. The result was similar to the 1000 mL L1 NO gas treatment in broccoli, which delayed yellowing and retarded the degradation of Chl (Eum et al., 2009). Short term fumigation with NO gas effectively delayed yellowing and extended postharvest life of broccoli, as well as bean and bok choy (Soegiarto and Wills, 2004). NO can also act as a protector against various stressful impacts, scavenge free oxygen radicals and counteract oxidative damage by regulating cellular redox balance and accelerating the transformation of superoxide anion (O2) and enhancing the activities of antioxidant enzymes (Shi et al., 2007). SNP treatment reduced pericarp browning and preserved bioactive antioxidants in litchi (Barman et al., 2014), and retarded pericarp reddening of tomato fruit via improving the activities of SOD, CAT and POD (Lai et al., 2011). NO treatment enhanced chilling tolerance of banana fruit via improving the activities of antioxidant enzymes (Wu et al., 2014), and decreased lipid peroxidation in broccoli (Eum et al., 2009). More recently, Iakimova and Woltering (2015) found that prevents wound-induced browning and delays senescence through inhibition of hydrogen peroxide accumulation in freshcut lettuce. Du et al. (2015) reported that NO enhanced tolerance to salt stress and improved nutritional quality in spinach, by elevation of antioxidant enzyme activity of POD, SOD and CAT and thus alleviation of salt-induced oxidative damage. The present study firstly indicated that SNP treatment delayed the yellowing of broccoli florets via improving the activities of antioxidant enzymes of CAT, POD, and APX (Fig. 4). Yellowing is a characterization of chlorophyll degradation on the surface of broccoli florets. A reduction in the content of chlorophyll also occurred in all florets during storage. However, SNP-treated florets maintained higher level of chlorophyll than the control florets (Fig. 3). An increase of chlorophyll in potato and lettuce was also stimulated by NO treatment (Beligni and Lamattina, 2000). More recently, Wang et al. (2015) reported that SNP delayed chlorophyll degradation and enhanced antioxidant activity in banana fruits after cold storage. The degradation of chlorophyll was catalysed by several enzymes. Chlase is the first enzyme in the chlorophyll catabolic pathway and catalyzes the Chl a to produce Chlide a (Harpaz-Saad et al., 2007). Chl a can be degraded by Chl-POX in vitro, producing OHChl a (Yamauchi et al., 2004). OHChl a, however, does not accumulate, it is only an intermediate. Then MD (Suzuki et al., 2005) or MDS (Kunieda et al., 2005) further catalyzes Chl degradation, by removing Mg2+ from Chlide a to produce Pheide. The next step in the Chl catabolic
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pathway includes a Phride a oxygenase (PaO) which converts Pheide a into a red Chl catabolite (Park et al., 2007). PPH is involved in Chl degradation and its expression is enhanced during leaf senescence. Schelbert et al. (2009) analyzed PPH activity and surprisingly reported that phein is the substrate for PPH and that both phein a and b were catalyzed to the respective phytol-free pigments, pheide a/b. The activities of Chl-degrading enzymes corresponded with changes in Chl content in broccoli (Costa et al., 2005; Aiamla-or et al., 2010). Results in this study demonstrated that pre-treatment of broccoli with 200 mmol L1 SNP significantly inhibited the level activities of chlorophyll-degrading enzymes (Fig. 5). These findings confirmed the previous report that NO reduced chlorophyll degradation in broccoli florets during senescence (Eum et al., 2009). There is evidence that NO affects the gene expression in horticultural crops such as cucumber (Dong et al., 2012), banana (Wu et al., 2014), and tomato (Lai et al., 2011). The degradation of chlorophyll is a key factor for senescence of broccoli and the expression of relative genes has been analyzed. It was reported that UV-B treatment of broccoli reduced the expression level of BoCLH1, but did not reduce the expression level of BoCLH2 or BoCLH3 (Aiamla-or et al., 2012). Over expression of a PAO gene has been shown to produce a stay-green phenotype when senescence is induced in permanent darkness (Pružinská et al., 2003). In the present study, it was found that the expression level of BoCLH1, BoCLH2 and BoPAO, but not BoCLH3 of broccoli were reduced by the SNP treatment (Fig. 6) and the overall level of Chlase was also inhibited (Fig. 5A). In conclusion, the results indicate that an exogenous application of SNP effectively delayed chlorophyll degradation and the yellowing of broccoli florets during storage at 20 C. The mechanism by which SNP accomplished this may be through improving the activities of antioxidant enzymes, inhibiting the activity of Chl-degrading enzymes and related gene expression. However, improper amount of SNP may have some potential pharmacological effects, such as induction of apoptosis in normal oral epithelial cells (Brady et al., 2008) and promotion of cyanideinduced toxicity (Hirai et al., 2013). Therefore, prior to practical application of SNP to horticultural products, it is necessary to take the food safety issue into consideration. Acknowledgements This research was supported by Ministry of Agriculture of China (CARS-22-E-01 and 201203095), National Natural Science Foundation of China (31101364) and Beijing Academy of Agriculture and Forestry Sciences (CXJJ201304 and QNjj201404). References Aiamla-or, S., Yamauchi, N., Takino, S., Shigyo, M., 2009. Effect of UV-A and UV-B irradiation on broccoli (Brassica oleracea L.: Italica Group) floret yellowing during storage. Postharvest Biol. Technol. 54, 177–179. Aiamla-or, S., Kaewsuksaeng, S., Shigyo, M., Yamauchi, N., 2010. Impact of UV-B irradiation on chlorophyll degradation and chlorophyll-degrading enzyme activities in stored broccoli (Brassica oleracea L. Italica Group) florets. Food Chem. 120, 645–651. Aiamla-or, S., Nakajima, T., Shigyo, M., Yamauchi, N., 2012. Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets. Postharvest Biol. Technol. 63, 60–66. Asumi, F., Yasuo, S., Hirofumi, T., Yamauchi, N., 2010. Effects of postharvest ethanol vapor treatment on activities and gene expression of chlorophyll catabolic enzymes in broccoli florets. Postharvest Biol. Technol. 55, 97–102. Azevedo, M.M., Carvalho, A., Pascoal, C., Rodrigues, F., Cássioo, F., 2007. Responses of antioxidant defenses to Cu and Zn stress in two aquatic fungi. Sci. Total Environ. 377, 233–243. Bai, L.P., Sui, F.G., Ge, T.D., Sun, Z.H., Lu, Y.Y., Zhou, G.S., 2006. Effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of maize. Pedosphere 16, 326–332.
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Barman, K., Siddiqui, M.W., Patel, V.B., Prasad, M., 2014. Nitric oxide reduces pericarp browning and preserves bioactive antioxidants in litchi. Sci. Hortic. 171, 71–77. Beligni, M.V., Lamattina, L., 2000. Nitric oxide stimulates seed germination and deetiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 210, 215–221. Brady, G., Crean, S.J., Lorenzon, A., Kapas, S., 2008. IGF-I protects human oral buccal mucosal epithelial cells from sodium nitroprusside-induced apoptosis via PI3kinase. Growth Horm. IGF Res. 18, 298–306. Costa, M.L., Civello, P.M., Chaves, A.R., Martínez, G.A., 2005. Effect of hot air treatments on senescence and quality parameters of harvested broccoli (Brassica oleracea L. var italica) head. J. Sci. Food Agric. 85, 1154–1160. Costa, M.L., Vicente, A.R., Civello, P.M., Chaves, A.R., Martínez, G.A., 2006. UV-C treatment delays postharvest senescence in broccoli florets. Postharvest Biol. Technol. 39, 204–210. Dong, J., Yu, Q., Lu, L., Xu, M., 2012. Effect of yeast saccharide treatment on nitric oxide accumulation and chilling injury in cucumber fruit during cold storage. Postharvest Biol. Technol. 68, 1–7. Du, S.T., Liu, Y., Zhang, P., Liu, H.J., Zhang, X.Q., Zhang, R.R., 2015. Atmospheric application of trace amounts of nitric oxide enhances tolerance to salt stress and improves nutritional quality in spinach (Spinacia oleracea L.). Food Chem. 173, 905–911. Eum, H.L., Hwang, D.K., Lee, S.K., 2009. Nitric oxide reduced chlorophyll degradation in broccoli (Brassica oleracea L.: var. italica) florets during senescence. Food Sci. Technol. Int. 15, 223–228. Funamoto, Y., Yamauchi, N., Shigyo, M., 2006. Control of isoperoxidases involved in chlorophyll degradation of stored broccoli (Brassica oleracea) florets by heat treatment. J. Plant Physiol. 163, 141–146. Forney, C.F., Song, J., Fan, L., Hildebrand, P.D., Jordan, M.A., 2003. Ozone and 1methylcyclopropene alter the postharvest quality of broccoli. J. Am. Soc. Hortic. Sci. 128, 403–408. Hansen, M.E., Sørensen, H., Cantwell, M., 2001. Changes in acetaldehyde, ethanol and amino acid concentration in broccoli florets during air and controlled atmosphere storage. Postharvest Biol. Technol. 22, 227–237. Harpaz-Saad, S., Azoulay, T., Arazi, T., Ben-Yaakov, E., Mett, A., Shiboleth, Y.M., Hörtensteiner, S., Gidoni, D., Gal-On, A., Goldschmidt, E.E., Eyal, Y., 2007. Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated. Plant Cell 19, 1007–1022. Hirai, D.M., Copp, S.W., Ferguson, S.K., Holdsworth, C.T., Musch, T.I., Poole, D.C., 2013. NO donor sodium nitroprusside: evaluation of skeletal muscle vascular and metabolic dysfunction. Microvasc. Res. 85, 104–111. Hörtensteiner, S., 2006. Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 57, 55–77. Iakimova, E.T., Woltering, E.J., 2015. Nitric oxide prevents wound-induced browning and delays senescence through inhibition of hydrogen peroxide accumulation in fresh-cut lettuce. Innov. Food Sci. Emerg. Technol. 30, 157–169. Jia, C.G., Xu, C.J., Wei, J., Yuan, J., Yuan, G.F., Wang, B.L., Wang, Q.M., 2009. Effect of modified atmosphere packaging on visual quality and glucosinolates of broccoli florets. Food Chem. 114, 28–37. Kunieda, T., Amano, T., Shioi, Y., 2005. Search for chlorophyll degradation enzyme, Mg-dechelatase, from extract of Chenopodium album with native and artificial substrates. Plant Sci. 169, 177–183. Lai, T.F., Wang, Y.Y., Li, B.Q., Qin, G.Z., Tian, S.P., 2011. Defense responses of tomato fruit to exogenous nitric oxide during postharvest storage. Postharvest Biol. Technol. 62, 127–132. Lamattina, L., Garcia-Matta, C., Graziano, M., Pagnussat, G., 2003. Nitric oxide: the versatility of an extensive signal molecule. Annu. Rev. Plant Biol. 54, 109–136. Lemoine, M.L., Chaves, A.R., Martínez, G.A., 2010. Influence of combined hot air and UV-C treatment on the antioxidant system of minimally processed broccoli (Brassica oleracea L. var. Italica). LWT Food Sci. Technol. 43, 1313–1319. Leshem, Y.Y., Wills, R.B.H., Ku, V.V.V., 1998. Evidence for the function of the free radical gas—nitric oxide (NO) as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiol. Biochem. 36, 825–833.
Lin, Y.C., Yang, L., Paul, M., Zu, Y.G., Tang, Z.H., 2013. Ethylene promotes germination of Arabidopsis seed under salinity by decreasing reactive oxygen species: evidence for the involvement of nitric oxide simulated by sodium nitroprusside. Plant Physiol. Biochem. 73, 211–218. Ognjanovi c, B.I., Markovi c, S.D., Ethordevi c, N.Z., Trbojevi c, I.S., Stajn, A.S., Saici c, Z.S., 2010. Cadmium-induces lipid peroxidation and changes in antioxidant defense system in the rat testes: protective role of coenzyme Q10 and vitamin E. Reprod. Toxicol. 29, 191–197. Page, T., Griffiths, G., Buchanan-Wollaston, V., 2001. Molecular and biochemical characterization of postharvest senescence in broccoli. Plant Physiol.125, 718–727. Park, S.Y., Yu, J.W., Park, J.S., Li, J., Yoo, S.C., Lee, N.Y., Lee, S.K., Jeong, S.W., Seo, H.S., Koh, H.J., Jeon, J.S., Park, Y.I., Paek, N.C., 2007. The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19, 1649–1664. Pružinská, A., Anders, I., Tanner, G., Roca, M., Hörtensteiner, S., 2003. Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron–sulfur protein, encoded by the accelerated cell death 1 gene. Proc. Natl. Acad. Sci. U. S. A. 100, 15259–15264. Rangkadilok, N., Tomkins, B., Nicolas, M.E., Premier, R.R., Bennett, R.N., Eagling, D.R., Taylor, P.W., 2002. The effect of postharvest and packaging treatments on glucoraphanin concentration in broccoli (Brassica oleracea var. italica). J. Agric. Food Chem. 50, 7386–7391. Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., Hörtensteiner, S., 2009. Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21, 767– 785. Shi, Q., Ding, F., Wang, X., Wei, M., 2007. Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. Plant Physiol. Biochem. 45, 542–550. Siddiqui, M.H., Al-Whaibi, M.H., Basalah, M.O., 2011. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248, 447–455. Soegiarto, L., Wills, R.B.H., 2004. Short term fumigation with nitric oxide gas in air to extend the postharvest life of broccoli, green bean, and bok choy. HortTechnology 14, 538–540. Sun, B., Yan, H.Z., Liu, N., Wei, J., Wang, Q.M., 2012. Effect of 1-MCP treatment on postharvest quality characters, antioxidants and glucosinolates of Chinese kale. Food Chem. 131, 519–526. Suzuki, T., Shioi, Y., 2002. Re-examination of Mg-dechelation reaction in the degradation of chlorophylls using chlorophyllin a as a substrate. Photosynth. Res. 74, 217–223. Suzuki, T., Kunieda, F.M., Murai, F., Morioka, S., Shioi, Y., 2005. Mg-dechelation activity in radish cotyledons with artificial and native substrates: Mgchlorophyllin a and chlorophyllide a. Plant Physiol. Biochem. 43, 459–464. Wang, Y., Luo, Z., Du, R., 2015. Nitric oxide delays chlorophyll degradation and enhances antioxidant activity in banana fruits after cold storage. Acta Physiol. Plant. 37, 74. Williams, D.J., Critchley, C., Pun, S., Nottingham, S., O’Hare, T.J., 2008. Epithiospecifier protein activity in broccoli: the link between terminal alkenyl glucosinolates and sulforaphane nitrile. Phytochemistry 69, 2765–2773. Wills, R.B.H., Pristijono, P., Golding, J.B., 2008. Browning on the surface of cut lettuce slices inhibited by short term exposure to nitric oxide (NO). Food Chem. 107, 1387–1392. Wu, B., Guo, Q., Li, Q.P., Ha, Y.M., Li, X.P., Chen, W.X., 2014. Impact of postharvest nitric oxide treatment on antioxidant enzymes and related genes in banana fruit in response to chilling tolerance. Postharvest Biol. Technol. 92, 157–163. Yamauchi, N., Funamoto, Y., Shigyo, M., 2004. Peroxidase-mediated chlorophyll degradation in horticultural crops. Phytochem. Rev. 3, 221–228. Yuan, G.F., Sun, B., Yuan, J., Wang, Q.M., 2010. Effect of 1-methylcyclopropene on shelf life, visual quality, antioxidant enzymes and health-promoting compounds in broccoli florets. Food Chem. 118, 774–781. Zhang, X.Y., Hu, C.G., Yao, J.L., 2010. Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance. J. Plant Physiol. 167, 88–94. Zeng, C.L., Liu, L., Xu, G.Q., 2011. The physiological responses of carnation cut flowers to exogenous nitric oxide. Sci. Hortic. 127, 424–430.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Exogenous sodium nitroprusside treatment of broccoli florets extends shelf life, enhances antioxidant enzyme activity, and inhibits chlorophyll-degradation Junyan Shia,b , Lipu Gaoa,b , Jinhua Zuoa,b , Qing Wanga,b,* , Qian Wanga,b , Linlin Fana,b a
National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing Key Laboratory of Fruits and Vegetable Storage and Processing, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 100097, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 June 2015 Received in revised form 15 January 2016 Accepted 18 January 2016 Available online 8 February 2016
The effect of an exogenous application of sodium nitroprusside (SNP) on the shelf life, antioxidant enzyme and chlorophyll-degrading enzyme activity, and chlorophyll-degradation related gene expression was investigated in stored broccoli. The preliminary results indicated that the 200 mmol L1 SNP treatment had the greatest effect on extending shelf life of broccoli florets so this concentration was used in the remainder of the study. The SNP treatment delayed chlorophyll degradation, thus color was maintained and shelf-life was extended. The activity of the antioxidant enzymes, catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) was all enhanced while glutathione reductase (GR) activity was inhibited throughout the storage. Relative to the untreated control, the SNP treatment suppressed the activity of the chlorophyll-degrading enzymes, chlorophyllase (Chlase), chlorophylldegrading peroxidase (Chl-POX), Mg-dechelatase (MD), and pheophytinase (PPH), and also suppressed chlorophyllase I (BoCHL1), chlorophyllase II (BoCHL2), chlorophyllase III (BoCLH3) and pheophorbide a oxygenase (BoPAO) gene expression during the entire storage period. In summary, 200 mmol L1 SNP treatment of broccoli extends shelf life, enhances oxidative stress tolerance by enhancing the activity of antioxidant enzymes, and inhibits the activity of chlorophyll-degrading enzymes and related gene expression. The combined effect delayed the yellowing of broccoli florets by inhibiting chlorophyll degradation, thus extending the shelf life of broccoli florets. ã 2016 Published by Elsevier B.V.
Keywords: Sodium nitroprusside Broccoli Antioxidant enzyme activity Chlorophyll-degrading enzyme activity Gene expression
1. Introduction Broccoli (Brassica oleracea var. Italica), a cruciferous vegetable, contains a high level of antioxidant compounds. It is also rich in vitamins, and anti-carcinogenic compounds, such as glucosinolates, and sulforaphane nitrile (Williams et al., 2008). However, it is a highly perishable product, and florets undergo rapid yellowing at room temperature after harvest (Hansen et al., 2001), thus reducing the shelf life of the florets and resulting in a low-quality product (Funamoto et al., 2006). The yellowing of broccoli florets, the symptom of postharvest senescence, is accompanied by a loss in fresh weight, chlorophyll content, sugars, and proteins (Costa
* Corresponding author at: National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China. E-mail address: [email protected] (Q. Wang). http://dx.doi.org/10.1016/j.postharvbio.2016.01.007 0925-5214/ ã 2016 Published by Elsevier B.V.
et al., 2005). There is also a loss in aroma, a decrease in amino acid content (Hansen et al., 2001), a decrease in total aliphatic and indole glucosinolates, and a general reduction in antioxidant compounds (Jia et al., 2009). Additionally, lipid peroxidation and protein degradation increased (Page et al., 2001). Oxidative processes play an important role in plant senescence, thus antioxidant enzymes, such as catalases (CAT), superoxide dismutases (SOD), peroxidases (POD), and the ascorbate-glutathione, have evolved in plants to combat oxidative stress and delay senescence (Lemoine et al., 2010). Yellowing of broccoli florets results from a loss and degradation of chlorophyll (Chl). Chl a is degraded by enzymes that include chlorophyllase (Chlase) (Harpaz-Saad et al., 2007), chlorophylldegrading peroxidase (Yamauchi et al., 2004), Mg-dechelatase (MD) (Suzuki et al., 2005), or another Mg-dechelating substance (MDS), pheophorbide (pheide) a oxygenase (PaO), and red Chl
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catabolite reductase (Hörtensteiner, 2006). Chl degradation occurs mainly within the chloroplast. Moreover, the peroxidase-mediated Chl degradation may also occur in the vacuole (Yamauchi et al., 2004; Hörtensteiner, 2006). Several approaches have been used to delay the senescence of broccoli florets, including heat treatment (Lemoine et al., 2010), modified atmosphere packaging (Jia et al., 2009), UV irradiation (Costa et al., 2006; Aiamla-or et al., 2009, 2012), and chemical treatments such as ozone and 1-methylcyclopropene (1-MCP) (Forney et al., 2003). Nitric oxide (NO) is an important signaling molecule with diverse physiological functions for plants, participating in various cellular processes, such as respiratory metabolism, growth and development, as well as maturation and senescence (Lamattina et al., 2003). Some researchers found that NO gas or sodium nitroprusside (SNP, as a donor of exogenous NO) treatment, delayed ripening, senescence and enhanced the resistance in horticulture crops (Wills et al., 2008; Wu et al., 2014; Iakimova and Woltering, 2015). For instance, Leshem et al. (1998) originally reported that NO-treated broccoli markedly retained green color and firmness compared to the air control and the ethylene-treated broccoli. The senescence-delaying effects of NO were also observed in carnation, Geraldton wax flowers (Chamelaucium uncinatum Schauer) and waratah flowers (Telopea speciosissima, Proteaceae) (Zeng et al., 2011; Leshem et al., 1998). However, the metabolic and physiological responses of broccoli to NO treatment remains still poorly understood. The overall objective of present study was to determine the effect of NO treatment (via the exogenous application of SNP) on broccoli florets stored at 20 C. More specifically, the effects of SNP on broccoli floret quality parameters, antioxidant enzyme and chlorophyll-degrading enzyme activity, as well as chlorophylldegradation related gene expressions during storage were investigated. 2. Materials and methods 2.1. Plant material and treatments Broccoli (B. oleracea var. Italica, ‘Bao-shi’) was harvested from an organic vegetable farm in the town of Xiaotangshan, located in the Shunyi District of Beijing, China, top-iced and directly brought to the laboratory within 3 h. The selected broccoli ranged in diameter from 15 to 20 cm, were uniform in color, had no evidence of insect or disease injury, no mechanically-induced wounds, and all had tight-head clusters. The selected broccoli heads were randomly placed into five groups. One group was treated with distilled water (control) and four groups received exogenous application of 100, 200, 400, and 800 mmolL1 sodium nitroprusside (SNP). Broccoli heads were air dried and then placed in polyethylene film bags (0.03 mm in thickness), with the top folded over, placed in plastic crates, and stored in the dark at 20 1 C and 90% RH. An assessment of shelf life, and a visual color scale rating were made daily in replicates of nine broccoli heads per treatment. The florets, together with about 1 cm of stalk, were cut from the broccoli heads each day and served as a sampling unit. The samples were immediately frozen in liquid nitrogen and stored at 80 C. The frozen florets were used to determine chlorophyll content, antioxidant enzyme activity, chlorophyll-degrading enzyme activity, and the relative expression of genes coding for proteins involved in chlorophyll degradation. 2.2. Determination of shelf life The shelf life of broccoli florets was determined according to the previous study (Yuan et al., 2010). A percentage of 30% yellowing florets was designated as the end of shelf life.
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2.3. Measurement of visual color rating scale The color of broccoli florets was measured by a visual assessment of changes from green to yellow as previously described (Rangkadilok et al., 2002). A color rating scale from 0 to 9 was adopted to indicate the general percentage of yellowing in the broccoli head, where 0 indicates all dark green, 1 indicates 3– 5% yellowing, 5 indicates 50% yellowing of the head, 7 indicates 75% yellowing of the head, and 9 indicates 100% yellowing of the broccoli heads. 2.4. Total chlorophyll content Frozen floret tissues were homogenized in acetone:ethanol (2:1) using a mortar and pestle. The homogenate was then filtrated and the filtrate was used to measure chlorophyll content according to Sun et al. (2012). The optical density at 645 nm and 643 mm were recorded for each sample. 2.5. Antioxidant enzyme activity Three grams of frozen flower tissue was extracted with 15 mL 0.1 molL1 phosphate buffer (PBS, pH 7.8, containing 0.5% polyvinylpyrrolidone), then centrifuged at 12,000 g for 20 min at 4 C. The supernatant was collected and used for enzyme assay. Catalase (CAT) activity was measured by determining the decrease in absorbance at 240 nm due to H2O2 consumption as described by Azevedo et al. (2007). The reaction mixture contained: 1.0 mL of 0.3% H2O2, 1.9 mL of 0.05 molL1 phosphate buffer (PBS, pH 7.8), and 0.1 mL of sample. Peroxidase (POD) activity was measured according to Bai et al. (2006). One gram of frozen floret tissue was homogenized in 8.0 mL of 0.05 M PBS (pH 7.8). The reaction mixture contained: 1.0 mL of 0.3% H2O2, 1.0 mL of 0.05 M PBS (pH 7.8), 0.9 mL of 0.2% guaiacol, and 0.1 mL of sample. The increase in absorbance at 470 nm over one minute was determined. Glutathione reductase (GR) activity was assayed by measuring NADPH oxidation in the presence of oxidized glutathione (Ognjanovi c et al., 2010). The reaction mixture consisted of 2.7 mL PBS (pH 7.5, containing 1 mmol L1 EDTA), 0.1 mL of 5 mmol L1 oxidized glutathione, 0.2 mL of sample, and 40 mL of 4.0 mmol L1 NADPH. GR activity was determined as a change in absorbance at 340 nm over 1 min. Ascorbate peroxidase (APX) activity was determined by measuring the decrease in absorbance of ascorbic acid (AsA) at 290 nm (Zhang et al., 2010). The reaction mixture contained 2.6 mL of PBS (pH 7.5, containing 0.1 mmol L1 EDTA and 0.5 mmol L1 AsA), 0.1 mL of sample, and 0.3 mL of 2 mmol L1 H2O2. 2.6. Chlorophyll-degrading enzyme activity An acetone-derived powder of floral tissues was prepared by the method of Aiamla-or et al. (2012). About 1 g broccoli floral tissues was homogenized in cold acetone (20 C). The homogenate was placed at 20 C for 15 min, then centrifuged at 12,000 g for 5 min at 4 C. The precipitate was then washed twice with cold acetone. The resulting precipitate was used as the source of acetone powder in the following assays. Chl a was bought from sigma company, and then 200 mg mL1 Chl a was prepared in acetone. Chlorophyllin a (Chlin) was prepared with 30% KOH in methanol from chl a in petroleum ether. Pheophytin (Phy) a was prepared by the acidic reaction from Chl a, using 0.1 M hydrochloric acid. The Phy a concentration was measured spectrophotometrically at 409 nm (Aiamla-or et al., 2012). Chlorophyll-degrading enzymes were extracted and measured as described by Aiamla-or et al. (2010).
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Chlorophyllase (Chlase) activity was determined as described by Aiamla-or et al. (2010). The reaction mixture contained 0.5 mL PBS (pH 7.5), 0.2 mL of 200 mg mL1 Chl a acetone solution, and 0.5 mL of enzyme extract. The reaction mixture was incubated in a water bath at 25 C for 40 min. The enzyme reaction was then stopped by adding 4 mL of acetone to the reaction mixture. Chlorophyllide (Chlide) was separated from the reaction mixture by adding 4 mL of hexane. The lower phase contained Chlide a. The level of Chlide was determined by measuring absorbance at 667 nm per unit. Chlorophyll-degrading peroxidase (Chl-POX) activity was determined as described by Asumi et al. (2010). The reaction mixture contained 0.5 mL of enzyme solution, 0.2 mL of 80 mg mL1 Chl a acetone solution, 0.1 mL of 0.3% H2O2, 0.1 mL of 1.0% Triton-X 100, 0.1 mL of 800 mmolL p-coumaric acid, and 1.5 mL of 0.1 molL1 PBS (pH 5.5). Activity was determined by measuring the decrease of Chl a by measuring the change in absorbance at 668 nm per unit at 25 C. Mg-dechelatase (MD) activity was measured according to Suzuki and Shioi (2002), using Chlin. Pheophorbin a formation was determined by measuring absorbance at 686 nm (Costa et al., 2005). The reaction mixture, containing 0.75 mL of 50 mmol L1 Tris–HCl buffer (pH 8.0), 0.3 mL of Chlin, and 0.2 mL of enzyme solution, was incubated at 37 C for 10 min. MD activity was expressed as the increment change in OD at 686 nm per minute under the test conditions. Pheophytinase (PPH) activity was measured based on the methods described by Schelbert et al. (2009) and Aiamla-or et al. (2012). The reaction mixture contained 0.35 mL of enzyme solution, 75 mL of 2.0% Triton-X 100, 0.1 mL of Phy a (10 mmol L1), and 0.70 mL of 50 mmol L1 Tris–HCl (pH 8.0). The reaction was incubated in darkness for 90 min at 25 C. The reaction was stopped by addition of acetone to the reaction mixture. Pheophorbide (pheide) a was separated from Phy a by the addition of hexane to the reaction mixture. Pheide a concentration in the acetone layer was measured spectrophotometrically at 667 nm. 2.7. RNA extraction and RT-qPCR analysis The frozen broccoli frozen florets tissues were first ground into a powder in liquid nitrogen. Total RNA was extracted from the powdered tissue using Trizol reagent (Invitrogen) and used as a template for cDNA synthesis utilizing SuperScript1 RT (Invitrogen). The resulting cDNA was stored at 80 C and subsequently used as a template for PCR using Ex Taq1 polymerase (Invitrogen) and gene-specific primers. The relative expression of the following genes was analyzed by RT-qPCR: Actin (BoAct, Genbank No. AF044573); chlorophyllase I (BoCLH1, GenBank NO. AF337544), chlorophyllase II (BoCLH2, GenBank NO. AF337545; chlorophyllase III (BoCLH3, GenBank NO. AF337546); and a Phride a oxygenase (BoPAO, GenBank NO. AF337544). The cDNA was used as a template for RT-qPCR utilizing UltraSYBR Mixture (Kangwei) in a Roche LightCycler1 480 machine according to the manufacturer’s instructions. Gene-specific primes were designed using Primer Express 5.0 (Applied Biosystem). Actin was used as a reference gene for standardization of all the results Aiamla-or et al. (2012). The gene-specific primers used to amplify each gene were as follows: BoAct, forward 50 CTTGCAC CAAGCAGCATGAA-30 , reverse 50 -AGAATGGAACC ACCGATCCA-30 ; BoCLH1 forward 50 -CCCCGTCGTCTTATTCTT-3, reverse 50 -TGTCCCTTCGTAGCCTTA-30 ; BoCLH2 forward 50 -CCACAAGT AACACCCAACC-30 , reverse 50 -ACCTTTCCCTGTCCCATC-30 ; BoCLH3 forward 50 -CGGTGG AGGAAGGAGAATA-30 , reverse 50 TTGTGGTGGAAGAAA GTGG-30 ; and BoPAO forward 50 -TCGGAACGATCAGAAATG-30 , reverse 50 -AAGTAGCAGC- CTGTGGAA-30 . Each measurement was performed in triplicate.
2.8. Statistical analysis All statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data from each analysis was subjected to a one-way ANOVA and means separations were compared by LSD test at a significance level of P < 0.05. No significant interactions between the treatments and time of sampling were identified. Data are presented as means and standard deviations. 3. Results 3.1. Effect of SNP treatment on shelf life In this study, 800 mmolL1 SNP treatment significantly decreased the shelf life of broccoli florets, while the other concentrations of SNP extended shelf life (Fig. 1). The greatest increase in shelf life was observed in broccoli florets treated with 200 mmolL1 SNP, which extended the shelf life by one day relative to the untreated control. Based on these results, 200 mmol L1 SNP was selected for more detailed studies. 3.2. Effect of 200 mmolL1 SNP on yellowing The yellowing of broccoli, stored at 20 C rapidly increased over a four-day period (Fig. 2). The SNP treatment, however, significantly delayed the yellowing of broccoli florets. The delay of yellowing by the SNP treatment was clearly observed after two days of storage. 3.3. Chlorophyll content A significant decrease in total chlorophyll content was observed in broccoli florets during storage at 20 C in both groups (control and SNP) (Fig. 3). The level of decrease in chlorophyll content, however, was lower in SNP-treated broccoli florets relative to the control. This difference was evident by day 4 where the total chlorophyll content of the SNP-treated broccoli had decreased by 52.55% from the initial chlorophyll content, while the control exhibited a 74.95% decrease relative to the initial chlorophyll content. 3.4. Antioxidant enzyme activity A decrease in CAT activity of broccoli was observed during the first 2 d of storage at 20 C in the control and SNP-treated groups. This was followed by and increase during days 3 and 4 in both groups (Fig. 4A). CAT activity, however was significantly higher in
Fig. 1. Effect of different concentrations of sodium nitroprusside (SNP) on the shelf life of broccoli florets stored at 20 C. Each bar is the mean sd of three replicate samples, where each replicate consists of nine broccoli heads. Values not sharing a common letter are significantly different at p < 0.05.
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Fig. 2. Percent change in the color (yellowing) of broccoli florets in broccoli heads treated with water (control) or 200 mmolL1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
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(Fig. 4B). POD activity in SNP-treated samples was higher, relative to the control group, especially on days 3 and 4. Levels of POD activity were significantly higher on days 3 and 4 in the SNPtreated group than in the control group. A gradual decrease in GR activity was observed in the broccoli floret tissues of both SNP-treated and untreated, control samples during the 4 d storage period at 20 C (Fig. 4C). SNP treatment of broccoli, however, suppressed the decline in GR activity, except in the 1 d samples. A linear decline in APX activity was observed in both groups of broccoli florets (Fig. 4D), represented by a 46.85% and 21.42% decline, relative to the initial activity, in the control and SNP groups, respectively, over the 4 d period of storage at 20 C. At 4 d, APX activity greatly increased in SNP-treated samples while activity continued to decline in the control. 3.5. Chlorophyll-degrading enzyme activity
Fig. 3. Change in total chlorophyll content in broccoli florets in broccoli heads which had been treated with water (control) or 200 mmolL1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
the SNP group on day 3. No significant differences were observed on day 4. POD activity steadily increased in both the control and SNPtreated samples over the four day period of storage at 20 C
Chlase activity in florets of both the control and SNP-treated groups exhibited a significant decrease after one day at 20 C (Fig. 5A). At 2 d onward, however, the activity was lower in the SNPtreated group than in the control group. Chlase activity increased to 151.03 nmolg1h1 at day 2, from 140.91 nmolg1h1 on day 1, in the control samples, while the activity in SNP-treated florets continued to decrease. Chl-POX activity increased in both the control and the SNP treatment by day 2. After that, however, activity sharply increased in the untreated (control) broccoli but much less in the SNP-treated broccoli. Chl-POX activity remained high in the control group on days 3 and 4 but sharply decreased (day 3) and then increased (day 4) in the SNP-treated group. The level of activity in the SNP-treated groups was significantly lower than in the control group on day 4 (Fig. 5B). MD activity was examined by using Chlin as a substrate. MD activity in the two groups increased during the whole period of storage but increased to a greater extent in the untreated, control group of broccoli, relative to the SNP-treated samples of broccoli (Fig. 5C). The SNP treatment suppressed MD activity in broccoli
Fig. 4. Changes in CAT (A), POD (B), GR (C), and APX (D) activity in broccoli florets of broccoli heads treated with water (control) or 200 mmolL1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
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Fig. 5. Changes in Chlase (A), Chl-POX (B), MD (C), and PPH (D) activity in broccoli florets. Broccoli heads were treated with water (Control) or 200 mmol L1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
florets and delayed yellowing. Initial PPH activity at the time of harvest was approximately 3.86 nmolg1h1, but increased to 4.31 nmolg1h1 and 3.43 nmolg1h1 in control and SNPtreated samples, respectively, after 1 d (Fig. 5D). PPH activity in broccoli florets increased gradually in both groups but was significantly higher in the control group, except on day 4. 3.6. RT-qPCR analysis The relative expression of BoCLH1 gene in the control broccoli florets greatly increased after one day in storage, and then greatly
decreased (Fig. 6A). In contrast, BoCLH1 expression in the SNPtreated group increased only a small amount after 1 d in storage, then decreased during days 2 and 3, and finally increased slightly on day 4. Expression of BoCLH2 gene initially decreased on day 1 and then gradually increased during the remainder of the storage period (Fig. 6B). BoCLH2 expression in the SNP-treated group also exhibited a decrease after 1 day of storage, followed by a slight increase on day 2, a very sharp decrease on day 3, and then a significant increase on day 4, at which time the level of activity was similar in both groups.
Fig. 6. Changes in BoCLH1 (A), BoCLH2 (B), BoCLH3 (C), and BoPAO (D) relative gene expression in broccoli florets. Broccoli heads were treated with water (Control) or 200 mmol L1 sodium nitroprusside (SNP) and stored at 20 C for 4 d. Each data point is the mean sd of three replicate samples.
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The expression of BoCLH3 was relatively low in both groups during the first three days of storage but then sharply increased about 6-fold in the SNP-treated group while remaining low in the control group (Fig. 6C). The pattern of expression of BoPAO in the control and SNP-treated broccoli florets was similar over the storage period, except on day 3, at which time expression decreased in SNP-treated samples but remained the same in the control group. Levels of BoPAO expression on day 4 were similar in both the SNP-treated and untreated control samples (Fig. 6D). 4. Discussion Evidences for the signaling roles of NO in plants have been obtained by measuring endogenous NO content and physiological changes after treatment with NO or SNP (Lin et al., 2013; Siddiqui et al., 2011). It can inhibit ethylene biosynthesis, delay ripening and senescence and enhance disease resistance in horticultural plants, including broccoli (Leshem et al., 1998; Soegiarto and Wills, 2004; Eum et al., 2009). In the present study, treatment with 200 mmol L1 SNP was shown to be effective in delaying the senescence. Compared with untreated broccoli, the shelf life prolonged one day (Fig. 1), the yellowing were retarded (Fig. 2) stored at 20 C. The result was similar to the 1000 mL L1 NO gas treatment in broccoli, which delayed yellowing and retarded the degradation of Chl (Eum et al., 2009). Short term fumigation with NO gas effectively delayed yellowing and extended postharvest life of broccoli, as well as bean and bok choy (Soegiarto and Wills, 2004). NO can also act as a protector against various stressful impacts, scavenge free oxygen radicals and counteract oxidative damage by regulating cellular redox balance and accelerating the transformation of superoxide anion (O2) and enhancing the activities of antioxidant enzymes (Shi et al., 2007). SNP treatment reduced pericarp browning and preserved bioactive antioxidants in litchi (Barman et al., 2014), and retarded pericarp reddening of tomato fruit via improving the activities of SOD, CAT and POD (Lai et al., 2011). NO treatment enhanced chilling tolerance of banana fruit via improving the activities of antioxidant enzymes (Wu et al., 2014), and decreased lipid peroxidation in broccoli (Eum et al., 2009). More recently, Iakimova and Woltering (2015) found that prevents wound-induced browning and delays senescence through inhibition of hydrogen peroxide accumulation in freshcut lettuce. Du et al. (2015) reported that NO enhanced tolerance to salt stress and improved nutritional quality in spinach, by elevation of antioxidant enzyme activity of POD, SOD and CAT and thus alleviation of salt-induced oxidative damage. The present study firstly indicated that SNP treatment delayed the yellowing of broccoli florets via improving the activities of antioxidant enzymes of CAT, POD, and APX (Fig. 4). Yellowing is a characterization of chlorophyll degradation on the surface of broccoli florets. A reduction in the content of chlorophyll also occurred in all florets during storage. However, SNP-treated florets maintained higher level of chlorophyll than the control florets (Fig. 3). An increase of chlorophyll in potato and lettuce was also stimulated by NO treatment (Beligni and Lamattina, 2000). More recently, Wang et al. (2015) reported that SNP delayed chlorophyll degradation and enhanced antioxidant activity in banana fruits after cold storage. The degradation of chlorophyll was catalysed by several enzymes. Chlase is the first enzyme in the chlorophyll catabolic pathway and catalyzes the Chl a to produce Chlide a (Harpaz-Saad et al., 2007). Chl a can be degraded by Chl-POX in vitro, producing OHChl a (Yamauchi et al., 2004). OHChl a, however, does not accumulate, it is only an intermediate. Then MD (Suzuki et al., 2005) or MDS (Kunieda et al., 2005) further catalyzes Chl degradation, by removing Mg2+ from Chlide a to produce Pheide. The next step in the Chl catabolic
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pathway includes a Phride a oxygenase (PaO) which converts Pheide a into a red Chl catabolite (Park et al., 2007). PPH is involved in Chl degradation and its expression is enhanced during leaf senescence. Schelbert et al. (2009) analyzed PPH activity and surprisingly reported that phein is the substrate for PPH and that both phein a and b were catalyzed to the respective phytol-free pigments, pheide a/b. The activities of Chl-degrading enzymes corresponded with changes in Chl content in broccoli (Costa et al., 2005; Aiamla-or et al., 2010). Results in this study demonstrated that pre-treatment of broccoli with 200 mmol L1 SNP significantly inhibited the level activities of chlorophyll-degrading enzymes (Fig. 5). These findings confirmed the previous report that NO reduced chlorophyll degradation in broccoli florets during senescence (Eum et al., 2009). There is evidence that NO affects the gene expression in horticultural crops such as cucumber (Dong et al., 2012), banana (Wu et al., 2014), and tomato (Lai et al., 2011). The degradation of chlorophyll is a key factor for senescence of broccoli and the expression of relative genes has been analyzed. It was reported that UV-B treatment of broccoli reduced the expression level of BoCLH1, but did not reduce the expression level of BoCLH2 or BoCLH3 (Aiamla-or et al., 2012). Over expression of a PAO gene has been shown to produce a stay-green phenotype when senescence is induced in permanent darkness (Pružinská et al., 2003). In the present study, it was found that the expression level of BoCLH1, BoCLH2 and BoPAO, but not BoCLH3 of broccoli were reduced by the SNP treatment (Fig. 6) and the overall level of Chlase was also inhibited (Fig. 5A). In conclusion, the results indicate that an exogenous application of SNP effectively delayed chlorophyll degradation and the yellowing of broccoli florets during storage at 20 C. The mechanism by which SNP accomplished this may be through improving the activities of antioxidant enzymes, inhibiting the activity of Chl-degrading enzymes and related gene expression. However, improper amount of SNP may have some potential pharmacological effects, such as induction of apoptosis in normal oral epithelial cells (Brady et al., 2008) and promotion of cyanideinduced toxicity (Hirai et al., 2013). Therefore, prior to practical application of SNP to horticultural products, it is necessary to take the food safety issue into consideration. Acknowledgements This research was supported by Ministry of Agriculture of China (CARS-22-E-01 and 201203095), National Natural Science Foundation of China (31101364) and Beijing Academy of Agriculture and Forestry Sciences (CXJJ201304 and QNjj201404). References Aiamla-or, S., Yamauchi, N., Takino, S., Shigyo, M., 2009. Effect of UV-A and UV-B irradiation on broccoli (Brassica oleracea L.: Italica Group) floret yellowing during storage. Postharvest Biol. Technol. 54, 177–179. Aiamla-or, S., Kaewsuksaeng, S., Shigyo, M., Yamauchi, N., 2010. Impact of UV-B irradiation on chlorophyll degradation and chlorophyll-degrading enzyme activities in stored broccoli (Brassica oleracea L. Italica Group) florets. Food Chem. 120, 645–651. Aiamla-or, S., Nakajima, T., Shigyo, M., Yamauchi, N., 2012. Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets. Postharvest Biol. Technol. 63, 60–66. Asumi, F., Yasuo, S., Hirofumi, T., Yamauchi, N., 2010. Effects of postharvest ethanol vapor treatment on activities and gene expression of chlorophyll catabolic enzymes in broccoli florets. Postharvest Biol. Technol. 55, 97–102. Azevedo, M.M., Carvalho, A., Pascoal, C., Rodrigues, F., Cássioo, F., 2007. Responses of antioxidant defenses to Cu and Zn stress in two aquatic fungi. Sci. Total Environ. 377, 233–243. Bai, L.P., Sui, F.G., Ge, T.D., Sun, Z.H., Lu, Y.Y., Zhou, G.S., 2006. Effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of maize. Pedosphere 16, 326–332.
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Barman, K., Siddiqui, M.W., Patel, V.B., Prasad, M., 2014. Nitric oxide reduces pericarp browning and preserves bioactive antioxidants in litchi. Sci. Hortic. 171, 71–77. Beligni, M.V., Lamattina, L., 2000. Nitric oxide stimulates seed germination and deetiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 210, 215–221. Brady, G., Crean, S.J., Lorenzon, A., Kapas, S., 2008. IGF-I protects human oral buccal mucosal epithelial cells from sodium nitroprusside-induced apoptosis via PI3kinase. Growth Horm. IGF Res. 18, 298–306. Costa, M.L., Civello, P.M., Chaves, A.R., Martínez, G.A., 2005. Effect of hot air treatments on senescence and quality parameters of harvested broccoli (Brassica oleracea L. var italica) head. J. Sci. Food Agric. 85, 1154–1160. Costa, M.L., Vicente, A.R., Civello, P.M., Chaves, A.R., Martínez, G.A., 2006. UV-C treatment delays postharvest senescence in broccoli florets. Postharvest Biol. Technol. 39, 204–210. Dong, J., Yu, Q., Lu, L., Xu, M., 2012. Effect of yeast saccharide treatment on nitric oxide accumulation and chilling injury in cucumber fruit during cold storage. Postharvest Biol. Technol. 68, 1–7. Du, S.T., Liu, Y., Zhang, P., Liu, H.J., Zhang, X.Q., Zhang, R.R., 2015. Atmospheric application of trace amounts of nitric oxide enhances tolerance to salt stress and improves nutritional quality in spinach (Spinacia oleracea L.). Food Chem. 173, 905–911. Eum, H.L., Hwang, D.K., Lee, S.K., 2009. Nitric oxide reduced chlorophyll degradation in broccoli (Brassica oleracea L.: var. italica) florets during senescence. Food Sci. Technol. Int. 15, 223–228. Funamoto, Y., Yamauchi, N., Shigyo, M., 2006. Control of isoperoxidases involved in chlorophyll degradation of stored broccoli (Brassica oleracea) florets by heat treatment. J. Plant Physiol. 163, 141–146. Forney, C.F., Song, J., Fan, L., Hildebrand, P.D., Jordan, M.A., 2003. Ozone and 1methylcyclopropene alter the postharvest quality of broccoli. J. Am. Soc. Hortic. Sci. 128, 403–408. Hansen, M.E., Sørensen, H., Cantwell, M., 2001. Changes in acetaldehyde, ethanol and amino acid concentration in broccoli florets during air and controlled atmosphere storage. Postharvest Biol. Technol. 22, 227–237. Harpaz-Saad, S., Azoulay, T., Arazi, T., Ben-Yaakov, E., Mett, A., Shiboleth, Y.M., Hörtensteiner, S., Gidoni, D., Gal-On, A., Goldschmidt, E.E., Eyal, Y., 2007. Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated. Plant Cell 19, 1007–1022. Hirai, D.M., Copp, S.W., Ferguson, S.K., Holdsworth, C.T., Musch, T.I., Poole, D.C., 2013. NO donor sodium nitroprusside: evaluation of skeletal muscle vascular and metabolic dysfunction. Microvasc. Res. 85, 104–111. Hörtensteiner, S., 2006. Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 57, 55–77. Iakimova, E.T., Woltering, E.J., 2015. Nitric oxide prevents wound-induced browning and delays senescence through inhibition of hydrogen peroxide accumulation in fresh-cut lettuce. Innov. Food Sci. Emerg. Technol. 30, 157–169. Jia, C.G., Xu, C.J., Wei, J., Yuan, J., Yuan, G.F., Wang, B.L., Wang, Q.M., 2009. Effect of modified atmosphere packaging on visual quality and glucosinolates of broccoli florets. Food Chem. 114, 28–37. Kunieda, T., Amano, T., Shioi, Y., 2005. Search for chlorophyll degradation enzyme, Mg-dechelatase, from extract of Chenopodium album with native and artificial substrates. Plant Sci. 169, 177–183. Lai, T.F., Wang, Y.Y., Li, B.Q., Qin, G.Z., Tian, S.P., 2011. Defense responses of tomato fruit to exogenous nitric oxide during postharvest storage. Postharvest Biol. Technol. 62, 127–132. Lamattina, L., Garcia-Matta, C., Graziano, M., Pagnussat, G., 2003. Nitric oxide: the versatility of an extensive signal molecule. Annu. Rev. Plant Biol. 54, 109–136. Lemoine, M.L., Chaves, A.R., Martínez, G.A., 2010. Influence of combined hot air and UV-C treatment on the antioxidant system of minimally processed broccoli (Brassica oleracea L. var. Italica). LWT Food Sci. Technol. 43, 1313–1319. Leshem, Y.Y., Wills, R.B.H., Ku, V.V.V., 1998. Evidence for the function of the free radical gas—nitric oxide (NO) as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiol. Biochem. 36, 825–833.
Lin, Y.C., Yang, L., Paul, M., Zu, Y.G., Tang, Z.H., 2013. Ethylene promotes germination of Arabidopsis seed under salinity by decreasing reactive oxygen species: evidence for the involvement of nitric oxide simulated by sodium nitroprusside. Plant Physiol. Biochem. 73, 211–218. Ognjanovi c, B.I., Markovi c, S.D., Ethordevi c, N.Z., Trbojevi c, I.S., Stajn, A.S., Saici c, Z.S., 2010. Cadmium-induces lipid peroxidation and changes in antioxidant defense system in the rat testes: protective role of coenzyme Q10 and vitamin E. Reprod. Toxicol. 29, 191–197. Page, T., Griffiths, G., Buchanan-Wollaston, V., 2001. Molecular and biochemical characterization of postharvest senescence in broccoli. Plant Physiol.125, 718–727. Park, S.Y., Yu, J.W., Park, J.S., Li, J., Yoo, S.C., Lee, N.Y., Lee, S.K., Jeong, S.W., Seo, H.S., Koh, H.J., Jeon, J.S., Park, Y.I., Paek, N.C., 2007. The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19, 1649–1664. Pružinská, A., Anders, I., Tanner, G., Roca, M., Hörtensteiner, S., 2003. Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron–sulfur protein, encoded by the accelerated cell death 1 gene. Proc. Natl. Acad. Sci. U. S. A. 100, 15259–15264. Rangkadilok, N., Tomkins, B., Nicolas, M.E., Premier, R.R., Bennett, R.N., Eagling, D.R., Taylor, P.W., 2002. The effect of postharvest and packaging treatments on glucoraphanin concentration in broccoli (Brassica oleracea var. italica). J. Agric. Food Chem. 50, 7386–7391. Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., Hörtensteiner, S., 2009. Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21, 767– 785. Shi, Q., Ding, F., Wang, X., Wei, M., 2007. Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. Plant Physiol. Biochem. 45, 542–550. Siddiqui, M.H., Al-Whaibi, M.H., Basalah, M.O., 2011. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248, 447–455. Soegiarto, L., Wills, R.B.H., 2004. Short term fumigation with nitric oxide gas in air to extend the postharvest life of broccoli, green bean, and bok choy. HortTechnology 14, 538–540. Sun, B., Yan, H.Z., Liu, N., Wei, J., Wang, Q.M., 2012. Effect of 1-MCP treatment on postharvest quality characters, antioxidants and glucosinolates of Chinese kale. Food Chem. 131, 519–526. Suzuki, T., Shioi, Y., 2002. Re-examination of Mg-dechelation reaction in the degradation of chlorophylls using chlorophyllin a as a substrate. Photosynth. Res. 74, 217–223. Suzuki, T., Kunieda, F.M., Murai, F., Morioka, S., Shioi, Y., 2005. Mg-dechelation activity in radish cotyledons with artificial and native substrates: Mgchlorophyllin a and chlorophyllide a. Plant Physiol. Biochem. 43, 459–464. Wang, Y., Luo, Z., Du, R., 2015. Nitric oxide delays chlorophyll degradation and enhances antioxidant activity in banana fruits after cold storage. Acta Physiol. Plant. 37, 74. Williams, D.J., Critchley, C., Pun, S., Nottingham, S., O’Hare, T.J., 2008. Epithiospecifier protein activity in broccoli: the link between terminal alkenyl glucosinolates and sulforaphane nitrile. Phytochemistry 69, 2765–2773. Wills, R.B.H., Pristijono, P., Golding, J.B., 2008. Browning on the surface of cut lettuce slices inhibited by short term exposure to nitric oxide (NO). Food Chem. 107, 1387–1392. Wu, B., Guo, Q., Li, Q.P., Ha, Y.M., Li, X.P., Chen, W.X., 2014. Impact of postharvest nitric oxide treatment on antioxidant enzymes and related genes in banana fruit in response to chilling tolerance. Postharvest Biol. Technol. 92, 157–163. Yamauchi, N., Funamoto, Y., Shigyo, M., 2004. Peroxidase-mediated chlorophyll degradation in horticultural crops. Phytochem. Rev. 3, 221–228. Yuan, G.F., Sun, B., Yuan, J., Wang, Q.M., 2010. Effect of 1-methylcyclopropene on shelf life, visual quality, antioxidant enzymes and health-promoting compounds in broccoli florets. Food Chem. 118, 774–781. Zhang, X.Y., Hu, C.G., Yao, J.L., 2010. Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance. J. Plant Physiol. 167, 88–94. Zeng, C.L., Liu, L., Xu, G.Q., 2011. The physiological responses of carnation cut flowers to exogenous nitric oxide. Sci. Hortic. 127, 424–430.