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The effect of temperature on phenolic content in wounded carrots
Food Chemistry 215 (2017) 116–123
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The effect of temperature on phenolic content in wounded carrots Cong Han a, Jing Li a, Peng Jin a, Xiaoan Li a, Lei Wang b, Yonghua Zheng a,⇑ a b
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China College of Agriculture, Liaocheng University, Liaocheng 252000, PR China
a r t i c l e
i n f o
Article history: Received 6 February 2016 Received in revised form 21 July 2016 Accepted 28 July 2016 Available online 29 July 2016 Chemical compounds studied in this article: Hydrogen peroxide (PubChem CID: 784) 2,2-Diphenyl-1-picrylhydrazyl (PubChem CID: 74358) Diphenyliodonium iodide (PubChem CID: 102228) Glucose (PubChem CID: 5793) Keywords: Temperature Phenolics Carrots Enzyme activities Antioxidant properties
a b s t r a c t Reactive oxygen species (ROS) have been shown to play important roles in biosynthesis of phenolic antioxidants in wounded carrots. This study has gone further to understand the effects of storage temperature on phenolics accumulation in wounded carrots. The results indicated that both increased wounding intensity and higher storage temperature promoted the generation of ROS and enhanced phenolics accumulation in wounded carrots. Moreover, treatment with ROS inhibitor inhibited ROS generation, suppressed the activities of key enzymes in phenylpropanoid pathway (phenylalanine ammonia lyase, PAL; cinnamate-4-hydroxylase, C4H; 4-coumarate coenzyme A ligase, 4CL) and restrained phenolics accumulation in shredded carrots confirming previous reports. In contrast, treatment with ROS elicitor promoted ROS generation, enhanced the activities of PAL, C4H and 4CL, and induced phenolics accumulation. Thus, our results confirmed that ROS are essential for mediating wound-induced phenolics accumulation in carrots and suggested that increase temperature enhanced the accumulation of phenolics through inducing ROS generation. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Wounding is one of the primary stresses experienced by freshcut produce, which will cause some physiological and biochemical changes to the tissue (Hodges & Toivonen, 2008). When wounding stress occurs, plants can adjust their metabolism to heal the damaged tissues and activate defense mechanisms that prevent further damage (León, Rojo, & Sánchez-Serrano, 2001). As part of defenserelated responses, many phenylpropanoid compounds are synthesized by triggering phenylalanine ammonium lyase (PAL) activity. The phenomena of wound-induced accumulation of phenolic compounds have been reported in many fruits and vegetables, including lettuce (Campos-Vargas & Saltveit, 2002), lemon (ArtésHernández, Rivera-Cabrera, & Kader, 2007), carrot (Surjadinata & Cisneros-Zevallos, 2012), and potato (Torres-Contreras, Nair, Cisneros-Zevallos, & Jacobo-Velázquez, 2014). Thus, wounding has been suggested as an innovative and simple tool to produce more phenolic compounds in horticultural crops (JacoboVelázquez & Cisneros-Zevallos, 2012). Among these products, ⇑ Corresponding author. E-mail address: [email protected] (Y. Zheng). http://dx.doi.org/10.1016/j.foodchem.2016.07.172 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
carrot is receiving considerable attention because of its remarkable accumulation of phenolic compounds after wounding and has been used as an ideal material to study the effect of additional stresses on wound-induced production of phenolic compounds and to elucidate the possible regulation mechanisms (Becerra-Moreno et al., 2015; Jacobo-Velázquez & Cisneros-Zevallos, 2012). Since Babic, Amiot, Nguyen-The, and Aubert (1993) first reported that chlorogenic acid accumulated in shredded carrots during storage, studies on the regulation of phenolic content in carrot tissues have been continued in the past two decades. For instance, Surjadinata and Cisneros-Zevallos (2012) quantitatively compared the total soluble phenolic (TSP) content of slices, pies, and shreds, and found that increasing wounding intensity was a feasible method to enhance the biosynthesis of phenolic antioxidants in carrots. Besides, wounding in combination with other postharvest abiotic stresses, such as hormone, controlled atmospheres, heat shock, hyperoxia stress, UV radiation, herbicide and water stress, can synergistically increase the accumulation of phenolic antioxidants in carrot (Alegria et al., 2012; Becerra-Moreno, Benavides, Cisneros-Zevallos, & Jacobo-Velázquez, 2012; BecerraMoreno et al., 2015; Du, Avena-Bustillos, Breksa, & McHugh, 2012; Heredia & Cisneros-Zevallos, 2009; Jacobo-Velázquez &
C. Han et al. / Food Chemistry 215 (2017) 116–123
Cisneros-Zevallos, 2012; Jacobo-Velázquez, Martinez-Hernandez, Rodriguez, Cao, & Cisneros-Zevallos, 2011; Simões, Allende, Tudela, Puschmann, & Gil, 2011). However, little is known about the effect of storage temperature on phenolics accumulation in wounded carrots. Reactive oxygen species (ROS), mainly superoxide radicals (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), are constantly generated in plant tissues in response to wounding and have been suggested to play important roles in mediating defense-related responses (Orozco-Cárdenas & Ryan, 1999). It has been shown that a wide range of environmental stresses such as high and low temperature, drought, salinity, UV or ozone stress and pathogen infections, can all contribute to enhancing ROS generation in plants (Breusegem, Vranová, Dat, & Inzé, 2001). Jacobo-Velázquez et al. (2011) found that ROS can act as signaling-molecules for the wound-induced accumulation of phenolic compounds in carrot. More recently they demonstrated that ROS play the central role on the wound-induced accumulation of phenolic compounds in carrot, whereas ethylene and jasmonic acid are essential to modulate ROS levels (Jacobo-Velázquez, GonzálezAgüero, & Cisneros-Zevallos, 2015). However, the effect of temperature on ROS levels and their relation to phenolics accumulation in wounded carrots is still unclear. The major objective of this study was to understand the effect of storage temperature on phenolics accumulation in wounded carrots. Therefore, we applied three different cutting styles and three storage temperatures to investigate the influences of wounding and temperature on ROS production, TSP content and antioxidant activity of wounded carrots. 2. Materials and methods 2.1. Chemical reagent Methanol was purchased from Guangdong Guanghua Sci-Tech CO., Ltd. (Guangdong, China). Acetone was purchased from Shanghai Lingfeng Chemical Regent CO., Ltd. (Shanghai, China). Diphenyliodonium iodide (DPI) was purchased from Tokyo Chemical Industry CO., Ltd. (Tokyo, JAPAN). Nitrotetrazolium blue chloride (NBT) and L-Phenylalanine were purchased from Shanghai Ryon Biological Technology CO., Ltd. (Shanghai, China). Folin– Ciocalteu reagent, ascorbic acid and glucose oxidase were purchased from Beijing Solarbio Science & Technology CO., Ltd. (Beijing, China). Glucose, hydroxyammonium chloride, p-aminophenylsulfonic acid, and a-naphthylamine were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used in this study were obtained from Sigma– Aldrich (St. Louis, MO, USA). All chemicals were of reagent grade. 2.2. Plant material, treatment and storage Fresh carrots (Daucus carota L. cv. Sanhongliucun) were purchased from a local wholesale market in Nanjing, P.R. China. After transferred to the laboratory, all carrots were washed and selected for uniform size (20–24 cm in length, 2.5–3 cm in diameter of equator), color, firmness, shape and free from blemishes. The selected carrots were conditioned at 10 °C overnight prior to two independent experiments. In the first experiment, wounding treatment was carried out according to the method of Surjadinata and Cisneros-Zevallos (2012). Carrots from each group were cut into slices, pies and shreds to create different wounding intensities. The wounding intensity of each cut type was defined by the ratio of the new surface area (A) created by wounding in cm2 over the tissue weight (W) in g. The calculated wounding intensities (A/W) were 4.9, 6.4 and 18.5 cm2/g, respectively, for slices, pies, and shreds. Whole carrots with 0.00 cm2/g of A/W were used as controls. After
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wounding treatment, slices, pies, and shreds were placed into 15 cm 10 cm 4 cm rigid polypropylene containers and stored at 4, 10 and 20 °C with 85–90% relative humidity for 7, 4 and 2 days, respectively. During storage, samples were taken and frozen in liquid nitrogen, and stored at 80 °C until use. In the second experiment, the impact of ROS on wound-induced accumulation of phenolic compounds in carrots was conducted. Shredded carrots were selected and immersed in the following solutions for 3 min: water, 300 lM diphenyliodonium iodide (DPI, ROS inhibitor), 50 lM glucose, 50 lM glucose/glucose oxidase (G/GO, ROS elicitor). Each treatment was applied to three replications. The concentrations of the chemicals were chosen based on the literatures (Jacobo-Velázquez et al., 2011; OrozcoCárdenas, Narváez-Vásquez, & Ryan, 2001) and our preliminary experiments. After treatment, all samples were then air-dried for approximately 15 min and stored at 20 °C with 85–90% RH for 48 h. During storage, samples were taken every 12 h, frozen in liquid nitrogen and stored at 80 °C until use. 2.3. Measurements of O 2 and H2O2 O 2 production was measured following the method of Elstner (1976) with some modifications. Five grams of fresh tissue was ground in 20 mL of 50 mM phosphate buffer (pH 7.8). The homogenate was centrifuged at 10,000g for 20 min at 4 °C. A sample of the crude extract (1 mL) was added to 1 mL of 1 M hydroxyammonium chloride and incubated at 25 °C for 1 h. Then 2 mL ether was added to the incubation mixture in order to prevent the interference of pigment and the mixture was centrifuged at 10,000g for 5 min. After that 1 mL mixture from the water layer, 1 mL of 17 mM p-aminophenylsulfonic acid and 7 mM a-naphthylamine (dissolved in glacial acetic acid: H2O = 3:1) were added and the mixture incubated at 25 °C for a further 20 min, the absorbance was measured at 530 nm. O 2 production was calculated against the standard curve using sodium nitrite as a standard and expressed as nM [NO2] g1 min1. For analysis of H2O2 content, three grams of fresh tissue was ground in 10 mL of chilled acetone. The homogenate was centrifuged at 10,000g for 20 min at 4 °C. The supernatant was collected immediately for H2O2 analysis using the method of Patterson, Mackae, and Ferguson (1984). The content of H2O2 was expressed as lM g1. 2.4. Total soluble phenolics (TSP) content assay TSP content was assayed following the method of Slinkard and Singleton (1977) with some modifications. Frozen tissue samples (5 g) were extracted using a mortar and pestle with 25 mL of cold methanol. The homogenate was centrifuged at 13,000g for 15 min at 4 °C and the supernatant was used for the TSP content assay. A sample of the crude extract (500 lL) was added to 1.5 mL of distilled water and 1 mL of FolinCiocalteu reagent, after that 1 mL 7.5% (w/v) Na2CO3 solution was added. The mixtures were incubated at 25 °C for 2 h before reading at 765 nm. The TSP content was expressed as milligrams of gallic acid (GAE) per kilogram. 2.5. Measurement of antioxidant capacity Antioxidant capacity was measured using 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical-scavenging activity as reported by De Ancos, Sgroppo, Plaza, and Cano (2002) with some modifications. The same extracts prepared for TSP assay were used for determination of antioxidant capacity. An aliquot of 0.2 mL of the TSP extract was added to 2.8 mL of 0.12 mM DPPH solution (prepared with ethanol). After incubated at 25 °C for 30 min, the absorbance was measured at 525 nm, and 0.2 mL of 80% ethanol
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adding to 2.8 mL of DPPH reagent was used as the control solution. The results were expressed as % DPPH inhibition and calculated with the following equation:
% DPPH inhibition ¼ ½ðA0 A1 Þ=A0 100 with A0, the absorbance of the control; and A1, the absorbance of the sample. 2.6. Phenylpropanoid metabolism-related enzymes assays Phenylalanine ammonium lyase (PAL) activity was assayed following the method of Assis, Maldonado, Muñoz, Escribano, and Merodio (2001). Four grams of fresh tissue was homogenized with 20 mL of ice-cold 50 mM sodium borate buffer (pH 8.8, containing 5 mM b-mercaptoethanol, 2 mM EDTA and 40 g/L PVP) and ground at 4 °C. The homogenate was centrifuged at 13,000g for 20 min at 4 °C and the supernatant was used for the enzyme assay. One milliliter of crude enzyme extraction solution was incubated with 2 mL of sodium borate buffer (50 mM, pH 8.8, containing 5 M bmercaptoethanol and 2 mM EDTA) and 0.5 mL of L-phenylalanine (20 mM), for 60 min, at 37 °C. The reaction was stopped by adding 0.1 mL of 6 M HCl. One unit of PAL activity was equal to a change of 0.01 at 290 nm per min, and expressed as U mg1 protein. Cinnamate-4-hydroxylase (C4H) activity was determined according to the method of Lamb and Rubery (1975) with some modifications. Four grams of fresh tissue was homogenized with 20 mL of ice-cold 50 mM Tris-HCl buffer (pH 8.9; containing 15 mM b-mercaptoethanol, 4 mM MgCl2, 5 mM ascorbic acid, 10 lM Leupeptin, 1 mM PMSF, 0.15% w/v PVP and 10% glycerol) and ground at 4 °C. The homogenate was centrifuged at 13,000g for 20 min at 4 °C and the supernatant was used for the enzyme assay. The reaction mixture contained 2.5 mL of 50 mM Tris-HCl buffer (pH 8.9; containing 2 lM trans-cinnamic acid, 2 lM NADPNa2, 5 lM G-6-PNa2) and 0.5 mL crude enzyme. The mixture was blended and incubated at 25 °C for 30 min and the reaction was stopped by adding 100 lL of 6 M HCl. The absorbance was measured at 340 nm before and after incubation. One unit of C4H activity was equal to a change of 0.01 in absorbance per min, and expressed as U mg1 protein. 4-coumarate coenzyme A ligase (4CL) activity was assayed as described by Knobloch and Hahlbrock (1975) with some modifications. Four grams of fresh tissue was homogenized with 20 mL of ice-cold 50 mM Tris-HCl buffer (pH 8.9; containing 15 mM bmercaptoethanol, 4 mM MgCl2, 5 mM ascorbic acid, 10 lM leupeptin, 1 mM PMSF, 0.15% w/v PVP and 10% glycerol) and ground at 4 °C. The reaction mixture contained 2 mL of 5 mM MgCl2, 0.5 mL of 5 mM adenosine triphosphate (ATP), 0.05 mL of 0.4 mM coenzyme A (CoA), 0.05 mL of 0.6 mM p-cumaric acid and 0.5 mL crude enzyme. The mixture was blended and incubated at 40 °C for 10 min and the reaction was stopped by adding 100 lL of 6 M HCl. The absorbance was measured at 333 nm before and after incubation. One unit of 4CL activity was equal to a change of 0.01 in absorbance per min, and expressed as U mg1 protein. 2.7. Antioxidant enzymes assays Five grams of fresh tissue were homogenized with 25 mL of icecold extraction buffers containing 0.2 g of PVPP and ground at 4 °C. For superoxide dismutase (SOD), the extraction buffer was 100 mM sodium phosphate (pH 7.8). For catalase (CAT) and ascorbate peroxidase (APX), the extraction buffer was 100 mM sodium phosphate (pH 7.0) while the buffer of APX containing 0.1 mM EDTA, 1 mM ascorbic acid and 1% polyvinyl-pyrrolidone. SOD activity was determined by the method of Rao, Paliyath, and Ormrod (1996). SOD was assayed by mixing 1.75 mL of 50 mM phosphate buffer (pH 7.8), 0.3 mL of 130 mM methionine,
0.3 mL of 750 lM NBT, 0.3 mL of 20 lM riboflavin, 0.3 mL of 100 lM EDTA-Na2, and 0.05 mL enzyme extract. Riboflavin was added at the end, and the reaction mixture was shaken and immediately placed under 4000 lx fluorescent lamp. One unit of SOD activity was defined as a change of 1 per min in OD560 and results were expressed as U mg1 protein. CAT activity was analyzed based on Wang and Tian (2005) with certain modifications. The reaction mixture consisted of 1.4 mL sodium phosphate buffer (50 mM, pH 7.0), 1 mL H2O2 (40 mM) and 0.6 mL enzyme extract. H2O2 was added at the end. One unit of CAT activity was defined as a change of 0.01 per min in OD240 and results were expressed as U mg1 protein. APX activity was determined by the method of Nakano and Asada (1989). The reaction mixture consisted of 2 mL sodium phosphate buffer (50 mM, pH 7.0), 0.8 mL H2O2 (2 mM) and 0.2 mL enzyme extract. H2O2 was added at the end. One unit of APX was defined a change of 0.01 per min in OD290, and results were expressed as U mg1 protein. Protein content in crude enzyme extracts was measured according to Bradford (1976) using bovine serum albumin as a standard. 2.8. Statistical analysis Experiments were performed using a completely randomized design and each treatment was replicated three times. All data were expressed as the mean ± standard error (SE) of three replicates and subjected to statistical analysis with SPSS 13.0 (SPSS Inc., Chicago, Illinois, US). The data were analyzed by one-way analysis of variance (ANOVA). Mean separations were performed using Duncan’s multiple range test, and differences at p < 0.05 were considered to be significant. In each figure, data points carrying different letters for the same storage time indicate statistically significant differences. 3. Results 3.1. Effects of wounding intensity and storage temperature on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity of wounded carrots As shown in Table 1, the O 2 production in all three cutting styles of carrots changed slightly and remained at relatively low levels at 4 °C. However, a greater accumulation of H2O2 content was observed in shreds compared with slices and pies. Increased wounding intensity and higher storage temperature both increased the ROS production of wounded carrots. The average values of O 2 and H2O2 in shreds were much higher than those of other two cutting styles during storage at 10 °C and 20 °C. The changes of TSP content and DPPH radical scavenging activity in all samples increased continuously during storage. Increased wounding intensity and higher storage temperature both accelerated the increase of TSP content and DPPH radical scavenging activity in wounded carrots. At the end of storage, the maximum value of TSP content was obtained in shreds at 20 °C, which showed a 4.8-fold increase compared to day 0 (233.13 mg GAE/kg for control). Correspondingly, the highest value of DPPH radical scavenging activity was observed in shredded carrots at 20 °C, which was 6.5 times higher than its initial value. 3.2. Effects of DPI and G/GO treatments on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity of shredded carrots Shredded carrots stored at 20 °C were selected for the second experiment. As shown in Table 2, the O 2 production in H2O treated samples decreased during the first 12 h and then experienced a
Table 1 a Effects of wounding intensity and storage temperature on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity in wounded carrots. Storage time (days)
1 O min1) 2 production (nM [NO2] g
H2O2content (lM g1)
Slices
Pies
Shreds
Slices
Pies
Shreds
Slices
Pies
Shreds
Slices
Pies
Shreds
4 °C
0 1 3 5 7
1.34 ± 0.04abA 1.26 ± 0.03cB 1.38 ± 0.03aA 1.31 ± 0.04bcB 1.31 ± 0.01bcA
1.36 ± 0.03bA 1.45 ± 0.04aA 1.38 ± 0.01bA 1.44 ± 0.04aA 1.33 ± 0.03bA
1.40 ± 0.03abA 1.44 ± 0.06aA 1.21 ± 0.03 dB 1.36 ± 0.05bcAB 1.30 ± 0.04bcA
8.59 ± 0.07bA 8.13 ± 0.14cC 8.97 ± 0.06aB 7.61 ± 0.08dC 8.51 ± 0.03bB
8.56 ± 0.12bA 9.33 ± 0.06aB 8.71 ± 0.10bC 9.27 ± 0.11aB 7.50 ± 0.15cC
8.61 ± 0.05eA 10.25 ± 0.13dA 14.20 ± 0.09cA 15.05 ± 0.12aA 14.44 ± 0.16bA
234.42 ± 4.12cA 278.31 ± 6.20bA 284.28 ± 6.05bB 315.15 ± 7.57aB 302.55 ± 10.62aB
235.26 ± 5.41cA 266.16 ± 5.66bA 298.17 ± 7.22aB 300.81 ± 12.10aB 310.05 ± 13.84aB
233.13 ± 6.83dA 244.83 ± 10.35 dB 334.53 ± 8.96cA 360.84 ± 4.83bA 445.98 ± 9.33aA
12.16 ± 0.46dA 15.44 ± 1.20cA 15.86 ± 0.64bcA 19.75 ± 1.01aA 17.72 ± 1.52bB
12.27 ± 0.65dA 14.60 ± 1.04cA 16.30 ± 0.26bA 17.37 ± 0.92abB 18.66 ± 1.28aB
12.12 ± 0.82dA 13.43 ± 1.45dA 16.70 ± 0.71cA 20.13 ± 0.50bA 28.50 ± 0.88aA
10 °C
0 1 2 3 4
1.34 ± 0.04dA 1.59 ± 0.04cB 1.56 ± 0.05cB 1.91 ± 0.08aB 1.73 ± 0.05bC
1.36 ± 0.03cA 1.86 ± 0.03bA 1.88 ± 0.04abA 1.92 ± 0.11abB 1.99 ± 0.06aB
1.40 ± 0.03dA 1.47 ± 0.07cdC 1.59 ± 0.06cB 2.32 ± 0.09bA 2.70 ± 0.10aA
8.59 ± 0.07dA 9.08 ± 0.04cB 11.26 ± 0.09bB 9.57 ± 0.07cC 13.70 ± 0.13aB
8.56 ± 0.12eA 8.79 ± 0.06dC 9.27 ± 0.08cC 10.68 ± 0.10aB 9.94 ± 0.06bC
8.61 ± 0.05eA 10.28 ± 0.07dA 12.56 ± 0.05cA 18.77 ± 0.14bA 19.23 ± 0.18aA
234.42 ± 4.12dA 342.09 ± 16.59cA 457.68 ± 18.51bA 462.72 ± 9.92bB 493.74 ± 11.46aB
235.26 ± 5.41eA 341.40 ± 7.43dA 367.59 ± 5.14cB 419.37 ± 16.09bC 460.38 ± 23.30aB
233.13 ± 6.83eA 263.85 ± 12.06 dB 387.57 ± 13.73cB 535.35 ± 25.82bA 669.84 ± 14.71aA
12.16 ± 0.46dA 17.85 ± 1.43cA 30.13 ± 0.66bA 30.44 ± 1.81bB 34.50 ± 0.94aB
12.27 ± 0.65eA 17.28 ± 0.92dA 22.12 ± 1.40cB 26.18 ± 1.30bC 29.39 ± 1.96aC
12.12 ± 0.82dA 14.96 ± 0.65 dB 22.67 ± 3.22cB 37.61 ± 2.41bA 44.84 ± 2.77aA
20 °C
0 0.5 1 1.5 2
1.34 ± 0.04bA 1.32 ± 0.05bB 1.26 ± 0.04bB 1.04 ± 0.05cC 1.65 ± 0.08aB
1.36 ± 0.03cA 1.44 ± 0.04cAB 2.05 ± 0.05aA 1.62 ± 0.08bB 1.34 ± 0.09cC
1.40 ± 0.03dA 1.53 ± 0.10dA 1.88 ± 0.14cA 3.48 ± 0.11aA 2.64 ± 0.15bA
8.59 ± 0.07cA 7.37 ± 0.06dC 7.46 ± 0.11dC 9.68 ± 0.08bC 12.01 ± 0.07aC
8.56 ± 0.12eA 9.72 ± 0.07 dB 11.13 ± 0.10cB 13.53 ± 0.02bB 22.84 ± 0.11aB
8.61 ± 0.05eA 11.19 ± 0.12dA 17.55 ± 0.05cA 26.12 ± 0.13bA 33.44 ± 0.17aA
234.42 ± 4.12dA 273.36 ± 5.38cAB 286.95 ± 11.42cC 364.44 ± 22.01bC 390.42 ± 11.17aC
235.26 ± 5.41eA 284.67 ± 4.45dA 335.76 ± 7.93cB 425.43 ± 18.22bB 609.42 ± 15.56aB
233.13 ± 6.83eA 265.17 ± 9.02 dB 436.23 ± 20.70cA 797.43 ± 13.64bA 1128.72 ± 16.55aA
12.16 ± 0.46cA 15.17 ± 1.38bA 16.33 ± 1.52bC 21.68 ± 0.85aC 24.42 ± 1.84aC
12.27 ± 0.65eA 15.64 ± 1.82dA 19.85 ± 0.87cB 27.13 ± 1.26bB 40.30 ± 2.55aB
12.12 ± 0.82dA 14.08 ± 2.47dA 29.21 ± 1.64cA 53.72 ± 3.68bA 78.27 ± 2.40aA
TSP content (mg GAE/kg)
DPPH radical scavenging activity (%)
a Data are expressed as the mean ± SD (n = 3). Values in the same storage temperature with different letters were significantly different at p < 0.05. Lowercase letters represented significant difference among storage time factors, and capital letters represented significant difference among cutting style factors.
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Storage temperature
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Table 2 a Effects of DPI and G/GO treatments on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity in shredded carrots during 2 days storage at 20 °C. Storage time (hours) 0 12 24 36 48
Shreds-H2O 1.06 ± 0.04bcB 0.85 ± 0.02 dB 0.99 ± 0.03cA 1.08 ± 0.06bA 1.29 ± 0.05aA
Shreds-DPI 0.94 ± 0.02aC 0.62 ± 0.03dC 0.54 ± 0.01eB 0.72 ± 0.05cB 0.83 ± 0.04bC
Shreds-glucose 1.08 ± 0.03bAB 0.88 ± 0.01cB 1.02 ± 0.05bA 1.08 ± 0.02bA 1.32 ± 0.04aA
Shreds-G/GO 1.13 ± 0.04abA 1.05 ± 0.03cdA 1.00 ± 0.03dA 1.10 ± 0.02bcA 1.19 ± 0.06aB
H2O2 content (lM g1)
0 12 24 36 48
6.84 ± 0.06eA 7.53 ± 0.03 dB 8.24 ± 0.05cC 9.65 ± 0.11bA 10.26 ± 0.08aA
6.16 ± 0.05 dB 6.63 ± 0.12cC 7.02 ± 0.02aD 7.11 ± 0.06aC 6.88 ± 0.07bD
6.88 ± 0.04eA 7.60 ± 0.07 dB 8.35 ± 0.09cB 9.23 ± 0.05bB 9.97 ± 0.09aB
6.85 ± 0.07eA 10.13 ± 0.10aA 8.98 ± 0.04dA 9.27 ± 0.08cB 9.47 ± 0.03bC
TSP content (mg GAE/kg)
0 12 24 36 48
176.16 ± 3.42eA 210.37 ± 2.53dA 301.65 ± 5.05cB 354.50 ± 14.55bB 412.35 ± 16.86aB
175.92 ± 4.84dA 184.29 ± 5.3 dB 204.45 ± 6.90cC 227.94 ± 11.43bC 261.63 ± 8.23aC
177.55 ± 3.68eA 211.51 ± 6.56dA 303.96 ± 12.45cB 361.92 ± 10.33bB 418.14 ± 14.72aB
176.34 ± 2.01eA 219.23 ± 5.87dA 332.49 ± 10.6cA 417.48 ± 15.16bA 466.38 ± 16.21aA
DPPH radical scavenging activity (%)
0 12 24 36 48
9.31 ± 0.72eA 14.48 ± 0.83dA 18.30 ± 1.2cB 23.83 ± 1.70bB 27.56 ± 1.39aB
9.22 ± 0.56cA 10.12 ± 1.73cB 11.68 ± 1.63bcC 13.61 ± 1.52bC 16.54 ± 1.26aC
9.26 ± 0.36eA 14.73 ± 1.47dA 18.77 ± 1.44cB 24.02 ± 0.95bB 28.02 ± 0.56aB
9.64 ± 1.13eA 14.61 ± 0.65dA 21.48 ± 0.85cA 27.99 ± 1.24bA 31.45 ± 1.08aA
1 O min1) 2 production (nM [NO2] g
a Data are expressed as the mean ± SD (n = 3). Values with different letters were significantly different at p < 0.05. Lowercase letters represented significant difference among storage time factors, and capital letters represented significant difference among treatment factors.
gradual increase in subsequent storage period. DPI treatment markedly inhibited the production of O 2 , and the significant (p < 0.05) difference was observed throughout the whole storage period. Similarly, the lower level of H2O2 was also observed in DPI treated samples. In contrast, the use of G/GO improved the initial production of O 2 , and brought a 47.9% increase of H2O2 content at the 12 h compared to 0 h. The TSP content and DPPH radical scavenging activity increased gradually during storage, whereas, their initial value or rising trend was weakened compared with un-immersed shreds. DPI treatment significantly (p < 0.05) restrained the increase of TSP content and DPPH radical scavenging activity in shredded carrot. In contrast, G/GO treatment induced the increase of TSP content and DPPH radical scavenging activity, and the values of these two parameters were 15.4% and 16.5% higher than control at 36 h, respectively. 3.3. Effects of DPI and G/GO treatments on PAL, C4H and 4CL activities of shredded carrots As shown in Fig. 1A, PAL activity in control shreds had a great increase during the first 24 h, thereafter kept at a high level in the second day. Similar as the pattern of PAL activity, the activities of C4H and 4CL in control samples increased gradually during the first 36 h (Fig. 1B and C). DPI treatment significantly (p < 0.05) inhibited the increase the activities of PAL, C4H and 4CL during the entire storage time. In contrast, G/GO treatment enhanced the activity of PAL and the significant (p < 0.05) difference was observed at the 24 h compared to control samples. The C4H activity of G/GO treated shreds experienced a pronounced increase from the 12 h to 36 h during storage, while for 4CL, the obvious increase was observed between 24 h and 36 h. 3.4. Effects of DPI and G/GO treatments on SOD, CAT and APX activities of shredded carrots SOD activity in control samples decreased markedly during the first 12 h, and then experienced a gradual increase afterward (Fig. 2A). DPI treatment significantly (p < 0.05) inhibited the increase of SOD activity during the whole storage time. Conversely, G/GO treatment enhanced the activity of SOD at 12 h, while no
significant difference (p > 0.05) was observed in the following storage time. As shown in Fig. 2B, the CAT activity in control shreds increased steadily during 48 h of storage. However, no significant changes were observed in DPI treated samples. The use of G/GO could effectively induce the activity of CAT apart from the last 12 h of storage. Unlike with the change of CAT, the APX activity in control shreds increased at 12 h, then decreased slightly at 24 h, thereafter increased dramatically (Fig. 2C). G/GO treatment improved the APX activity during the first 24 h, however, a lower value was observed at later storage period. Although the APX activity in DPI treated samples was relatively lower than control samples, an obvious increase was also observed between 12 h and 24 h. 4. Discussion ROS are generated in plant tissue during plant growth and development and have been recognized as signal molecules in plant defense responses (Vranová, Inz´e, & Van Breusegem, 2002). Many postharvest abiotic stresses such as temperature, atmospheric composition, wounding and phytohormones can induce ROS generation which affects physiological, metabolic properties and quality of fruits and vegetables (Hodges, Lester, Munro, & Toivonen, 2004). The physiological role of ROS on the accumulation of phenolic antioxidants in wounded carrots was previously reported (Jacobo-Velázquez et al., 2011, 2015). This study has gone further to understand the effects of storage temperature on ROS levels and TSP content in wounded carrots. As shown in Table 1, increased wounding intensity enhanced the formation of O 2 , the generation of H2O2 and induced a higher accumulation of phenolics in shredded carrots. This result confirmed the previous reports that the biosynthesis of phenolics in wounded carrot increased with increasing wounding intensity (Du et al., 2012; Surjadinata & Cisneros-Zevallos, 2012) and that the wound-induced accumulation of phenolic compounds in carrots was associated with the increase of ROS levels (JacoboVelázquez et al., 2011). Moreover, we found that higher storage temperature promoted the production of ROS and caused more TSP content in wounded carrots, suggesting that the application of higher storage temperature may increase wound-induced phenolic accumulation through enhancing ROS levels. In plant cells,
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Fig. 1. Effects of DPI and G/GO treatments on activities of PAL (A), C4H (B), and 4CL (C) in shredded carrots during 2 days storage at 20 °C. Data are expressed as the mean ± SD (n = 3). Data points carrying different letters for the same storage time indicate statistically significant differences (p < 0.05).
Fig. 2. Effects of DPI and G/GO treatments on activities of SOD (A), CAT (B), and APX (C) in shredded carrots during 2 days storage at 20 °C. Data are expressed as the mean ± SD (n = 3). Data points carrying different letters for the same storage time indicate statistically significant differences (p < 0.05).
the mitochondrial electron transport chain is a major site of ROS production (Møller, 2001). Higher storage temperature may provoke the mitochondrial respiratory activity, thereby inducing the production of ROS and the biosynthesis of phenolic compounds in wounded carrots. As an important category of phytochemicals,
phenolic compounds have been documented the main contributor to the antioxidant capacity of plants (Scalbert, Johnson, & Saltmarsh, 2005). The observed increase of antioxidant capacity (Table 1) in wounded carrots can be attributed to their higher TSP content.
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In the second experiment, to confirm the vital role of ROS on wound-induced phenolics accumulation, shredded carrots were used to assay the impact of ROS inhibitor or elicitor on changes of TSP content. Our results showed that treatment with the NADPH oxidase inhibitor–DPI could significantly reduce the generation of O 2 and H2O2 and suppressed and the accumulation of phenolics in shredded carrots and (Table 2). This result was consistent with the previous study of Jacobo-Velázquez et al. (2011), who found that the accumulation of phenolic compounds was almost completely blocked in DPI-treated carrot shreds. Glucose oxidase (GO) is a flavoprotein which catalyses the oxidation of b-Dglucose to produce D-glucono-d-lactone and H2O2 (Bankar, Bule, Singhal, & Ananthanarayan, 2009). In the present study, treatment with G/GO significantly enhanced H2O2 content of shredded carrots and markedly increased TSP content after 24 h of storage (Table 2). These observations confirmed previous reports of Jacobo-Velázquez et al. (2011, 2015) that ROS played a key role as signaling molecules for the wound-induced accumulation of phenolic compounds in wounded carrots. PAL, C4H and 4CL are three pivotal enzymes that catalyse the synthesis of phenolic compounds in phenylpropanoid pathways in plants (Weisshaar & Jenkins, 1998). PAL catalyses the conversion of phenylalanine to trans-cinnamic acid, and then hydroxylated to p-coumaric acid in the presence of C4H. As the metabolism of phenolic compounds continues, p-coumaric is transformed to p-coumaroyl CoA by 4CL and finally formed to caffeoylquinic acids and its derivatives, which are the major increased phenolic profiles in fresh-cut carrot (Becerra-Moreno et al., 2012; Jacobo-Velázquez et al., 2015). Many studies have demonstrated that increases of PAL, C4H and 4CL activities would result in the accumulation of phenolic compounds (Chen et al., 2006; Liu et al., 2014). Our experiment found that the activity of PAL was inhibited by DPI treatment, while activated by G/GO treatment (Fig. 1A). Likewise, the activities of C4H and 4CL showed similar tendency with PAL activity and matched with the accumulation of TSP content (Fig. 1B and C). These results were consistent with the findings of Jacobo-Velázquez et al. (2015) on the expression of genes encoding those enzymes, suggesting that ROS can affect phenolics biosynthesis in wounded carrots by inducing phenylpropanoid pathway enzymes. Also, it was noteworthy that the ‘‘oxidative burst” preceded the peak of these three enzymes, which may indicate that there exists a signal transduction time for carrot cell to mobilize these phenylpropanoid pathway enzymes. In order to maintain the concentration of ROS at relatively low level, plants have evolved mechanisms to scavenge these toxic and reactive substances by antioxidant compounds (e.g., phenolic compounds) and by antioxidant enzymes such as SOD, CAT and APX. SOD, as the major O 2 scavenging enzyme, catalyses the disproportionation of O 2 radicals into H2O2 and O2, while both CAT and APX catalyze the degradation of H2O2 to H2O and O2. In the present work, the activities of SOD, CAT and APX were suppressed in DPI-treated shreds (Fig. 2) and matched with the lower levels of O 2 and H2O2. This result confirmed the previous work by Jacobo-Velázquez et al. (2011) on those same enzymes. As for G/GO-treated shreds, the activities of SOD, CAT and APX were significantly enhanced, however, the contents of ROS were maintained at high level. This result could be caused by the G/GO reaction that produces H2O2, which in turn induce antioxidant enzymes activity. Previous studies have also shown that pretreatment with H2O2 enhanced antioxidant enzymes activity, thus inducing protection from chilling-induced oxidative stress in maize and tobacco (Gechev et al., 2002; Prasad, Anderson, Martin, & Stewart, 1994). These results confirmed previous work of Jacobo-Velázquez et al. (2011, 2015) that antioxidant enzymes played important role on modulating ROS levels in wounded carrots.
5. Conclusions This study confirmed previous findings that increased wounding intensity induced the formation of O 2 , the generation of H2O2, and the accumulation of phenolic compounds in wounded carrots. Moreover, higher storage temperature strengthened ROS production and phenolics accumulation in wounded carrots through enhancing ROS levels. ROS inhibitor DPI inhibited ROS generation, suppressed the activities of PAL, C4H, 4CL and caused a lower accumulation of phenolic compounds in shredded carrots confirming previous reports. Whereas, ROS elicitor G/GO enhanced ROS generation, induced the activities of these enzymes and brought a higher accumulation of phenolics. Thus, our results confirmed that ROS are essential for mediating wound-induced phenolics accumulation in carrots and suggested that increase temperature enhanced the accumulation of phenolics. Acknowledgments This study was financially supported by the National Natural Science Foundation of China (No. 31471632). References Alegria, C., Pinheiro, J., Duthoit, M., Gonçalves, E. M., Moldão-Martins, M., & Abreu, M. (2012). Fresh-cut carrot (cv. Nantes) quality as affected by abiotic stress (heat shock and UV-C irradiation) pre-treatments. LWT – Food Science and Technology, 48(2), 197–203. Artés-Hernández, F., Rivera-Cabrera, F., & Kader, A. A. (2007). Quality retention and potential shelf-life of fresh-cut lemons as affected by cut type and temperature. Postharvest Biology Technology, 43(2), 245–254. Assis, J. S., Maldonado, R., Muñoz, T., Escribano, M. I., & Merodio, C. (2001). Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biology and Technology, 23(1), 33–39. Babic, I., Amiot, M. J., Nguyen-The, C., & Aubert, S. (1993). Changes in phenolic contents in fresh ready-to-use shredded carrots during storage. Journal of Food Science, 58(2), 351–356. Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase – an overview. Biotechnology Advances, 27(4), 489–501. Becerra-Moreno, A., Benavides, J., Cisneros-Zevallos, L., & Jacobo-Velázquez, D. A. (2012). Plants as biofactories: glyphosate-induced production of shikimic acid and phenolic antioxidants in wounded carrot tissue. Journal of Agricultural and Food Chemistry, 60(45), 11378–11386. Becerra-Moreno, A., Redondo-Gil, M., Benavides, J., Nair, V., Cisneros-Zevallos, L., & Jacobo-Velázquez, D. A. (2015). Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot. Frontiers in Plant Science, 6, 837. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72(1–2), 248–254. Breusegem, F. V., Vranová, E., Dat, J. F., & Inzé, D. (2001). The role of active oxygen species in plant signal transduction. Plant Science, 161(3), 405–414. Campos-Vargas, R., & Saltveit, M. E. (2002). Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce. Plant Physiology, 114(1), 73–84. Chen, J. Y., Wen, P. F., Kong, W. F., Pan, Q. H., Wan, S. B., & Huang, W. D. (2006). Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development. Journal of Plant Physiology, 163(2), 115–127. De Ancos, B., Sgroppo, S., Plaza, L., & Cano, M. P. (2002). Possible nutritional and health related value promotion in orange juice preserved by high-pressure treatment. Journal of Agricultural and Food Chemistry, 82(8), 790–796. Du, W. X., Avena-Bustillos, R. J., Breksa, A. P., III, & McHugh, T. H. (2012). Effect of UV-B light and different cutting styles on antioxidant enhancement of commercial fresh-cut carrot products. Food Chemistry, 134(4), 1862–1869. Elstner, E. F. (1976). Inhibition of nitrite formation hydroxylam–mouium–chloride: a simple assay for superoxide dismutase. Analytical Biochemistry, 70, 616–620. Gechev, T. S., Gadjev, I., Van Breusegem, F., Inzé, D., Dukiandjiev, S., Toneva, V., & Minkov, I. (2002). Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. Cellular and Molecular Life Sciences CMLS, 59(4), 708–714. Heredia, J. B., & Cisneros-Zevallos, L. (2009). The effect of exogenous ethylene and methyl jasmonate on pal activity, phenolic profiles and antioxidant capacity of carrots (Daucus carota) under different wounding intensities. Postharvest Biology Technology, 51(2), 242–249. Hodges, D. M., & Toivonen, P. M. A. (2008). Quality of fresh-cut fruits and vegetables as affected by exposure to abiotic stress. Postharvest Biology Technology, 48(2), 155–162.
C. Han et al. / Food Chemistry 215 (2017) 116–123 Hodges, D. M., Lester, G. E., Munro, K. D., & Toivonen, P. M. A. (2004). Oxidative stress: importance for postharvest quality. HortScience, 39(5), 924–929. Jacobo-Velázquez, D. A., & Cisneros-Zevallos, L. (2012). An alternative use of horticultural crops: stressed plants as biofactories of bioactive phenolic compounds. Agriculture, 2(3), 259–271. Jacobo-Velázquez, D. A., González-Agüero, M., & Cisneros-Zevallos, L. (2015). Crosstalk between signaling pathways: the link between plant secondary metabolite production and wounding stress response. Scientific Reports, 5, 8608. Jacobo-Velázquez, D. A., Martinez-Hernandez, G. B., Rodriguez, S., Cao, C. M., & Cisneros-Zevallos, L. (2011). Plants as biofactories: Physiological role of reactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress. Journal of Agricultural and Food Chemistry, 59(12), 6583–6593. Knobloch, K. H., & Hahlbrock, K. (1975). Isoenzyme of p-coumarate: CoA ligase from cell suspension cultures of Glycine max. European Journal of Biochemistry, 52(2), 311–320. Lamb, C. J., & Rubery, P. H. (1975). A spectrophotometric assay for trans-cinnamic acid 4-hydroxylase activity. Analytical Biochemistry, 68(2), 554–561. León, J., Rojo, E., & Sánchez-Serrano, J. J. (2001). Wound signaling in plants. Journal of Experimental Botany, 52, 1–9. Liu, Y. Y., Ge, Y. H., Bi, Y., Li, C. Y., Deng, H. W., & Dong, B. Y. (2014). Effect of postharvest acibenzolar-S-methyl dipping on phenylpropanoid pathway metabolism in muskmelon (Cucumis melo L.) fruits. Scientia Horticulturae, 168 (26), 113–119. Møller, I. M. (2001). Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annual Review of Plant Biology, 52(1), 561–591. Nakano, Y., & Asada, K. (1989). Hydrogen peroxide is scavenged by ascrobate specific peroxidase in spinach chloroplasts. Plant and Cell Physiology, 22(5), 867–880. Orozco-Cárdenas, M. L., & Ryan, C. (1999). Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proceedings of the National Academy of Science, 96(11), 6553–6557. Orozco-Cárdenas, M. L., Narváez-Vásquez, J., & Ryan, C. A. (2001). Hydrogen peroxide acts as a second messenger for the induction of defense genes in
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The effect of temperature on phenolic content in wounded carrots Cong Han a, Jing Li a, Peng Jin a, Xiaoan Li a, Lei Wang b, Yonghua Zheng a,⇑ a b
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China College of Agriculture, Liaocheng University, Liaocheng 252000, PR China
a r t i c l e
i n f o
Article history: Received 6 February 2016 Received in revised form 21 July 2016 Accepted 28 July 2016 Available online 29 July 2016 Chemical compounds studied in this article: Hydrogen peroxide (PubChem CID: 784) 2,2-Diphenyl-1-picrylhydrazyl (PubChem CID: 74358) Diphenyliodonium iodide (PubChem CID: 102228) Glucose (PubChem CID: 5793) Keywords: Temperature Phenolics Carrots Enzyme activities Antioxidant properties
a b s t r a c t Reactive oxygen species (ROS) have been shown to play important roles in biosynthesis of phenolic antioxidants in wounded carrots. This study has gone further to understand the effects of storage temperature on phenolics accumulation in wounded carrots. The results indicated that both increased wounding intensity and higher storage temperature promoted the generation of ROS and enhanced phenolics accumulation in wounded carrots. Moreover, treatment with ROS inhibitor inhibited ROS generation, suppressed the activities of key enzymes in phenylpropanoid pathway (phenylalanine ammonia lyase, PAL; cinnamate-4-hydroxylase, C4H; 4-coumarate coenzyme A ligase, 4CL) and restrained phenolics accumulation in shredded carrots confirming previous reports. In contrast, treatment with ROS elicitor promoted ROS generation, enhanced the activities of PAL, C4H and 4CL, and induced phenolics accumulation. Thus, our results confirmed that ROS are essential for mediating wound-induced phenolics accumulation in carrots and suggested that increase temperature enhanced the accumulation of phenolics through inducing ROS generation. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Wounding is one of the primary stresses experienced by freshcut produce, which will cause some physiological and biochemical changes to the tissue (Hodges & Toivonen, 2008). When wounding stress occurs, plants can adjust their metabolism to heal the damaged tissues and activate defense mechanisms that prevent further damage (León, Rojo, & Sánchez-Serrano, 2001). As part of defenserelated responses, many phenylpropanoid compounds are synthesized by triggering phenylalanine ammonium lyase (PAL) activity. The phenomena of wound-induced accumulation of phenolic compounds have been reported in many fruits and vegetables, including lettuce (Campos-Vargas & Saltveit, 2002), lemon (ArtésHernández, Rivera-Cabrera, & Kader, 2007), carrot (Surjadinata & Cisneros-Zevallos, 2012), and potato (Torres-Contreras, Nair, Cisneros-Zevallos, & Jacobo-Velázquez, 2014). Thus, wounding has been suggested as an innovative and simple tool to produce more phenolic compounds in horticultural crops (JacoboVelázquez & Cisneros-Zevallos, 2012). Among these products, ⇑ Corresponding author. E-mail address: [email protected] (Y. Zheng). http://dx.doi.org/10.1016/j.foodchem.2016.07.172 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
carrot is receiving considerable attention because of its remarkable accumulation of phenolic compounds after wounding and has been used as an ideal material to study the effect of additional stresses on wound-induced production of phenolic compounds and to elucidate the possible regulation mechanisms (Becerra-Moreno et al., 2015; Jacobo-Velázquez & Cisneros-Zevallos, 2012). Since Babic, Amiot, Nguyen-The, and Aubert (1993) first reported that chlorogenic acid accumulated in shredded carrots during storage, studies on the regulation of phenolic content in carrot tissues have been continued in the past two decades. For instance, Surjadinata and Cisneros-Zevallos (2012) quantitatively compared the total soluble phenolic (TSP) content of slices, pies, and shreds, and found that increasing wounding intensity was a feasible method to enhance the biosynthesis of phenolic antioxidants in carrots. Besides, wounding in combination with other postharvest abiotic stresses, such as hormone, controlled atmospheres, heat shock, hyperoxia stress, UV radiation, herbicide and water stress, can synergistically increase the accumulation of phenolic antioxidants in carrot (Alegria et al., 2012; Becerra-Moreno, Benavides, Cisneros-Zevallos, & Jacobo-Velázquez, 2012; BecerraMoreno et al., 2015; Du, Avena-Bustillos, Breksa, & McHugh, 2012; Heredia & Cisneros-Zevallos, 2009; Jacobo-Velázquez &
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Cisneros-Zevallos, 2012; Jacobo-Velázquez, Martinez-Hernandez, Rodriguez, Cao, & Cisneros-Zevallos, 2011; Simões, Allende, Tudela, Puschmann, & Gil, 2011). However, little is known about the effect of storage temperature on phenolics accumulation in wounded carrots. Reactive oxygen species (ROS), mainly superoxide radicals (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), are constantly generated in plant tissues in response to wounding and have been suggested to play important roles in mediating defense-related responses (Orozco-Cárdenas & Ryan, 1999). It has been shown that a wide range of environmental stresses such as high and low temperature, drought, salinity, UV or ozone stress and pathogen infections, can all contribute to enhancing ROS generation in plants (Breusegem, Vranová, Dat, & Inzé, 2001). Jacobo-Velázquez et al. (2011) found that ROS can act as signaling-molecules for the wound-induced accumulation of phenolic compounds in carrot. More recently they demonstrated that ROS play the central role on the wound-induced accumulation of phenolic compounds in carrot, whereas ethylene and jasmonic acid are essential to modulate ROS levels (Jacobo-Velázquez, GonzálezAgüero, & Cisneros-Zevallos, 2015). However, the effect of temperature on ROS levels and their relation to phenolics accumulation in wounded carrots is still unclear. The major objective of this study was to understand the effect of storage temperature on phenolics accumulation in wounded carrots. Therefore, we applied three different cutting styles and three storage temperatures to investigate the influences of wounding and temperature on ROS production, TSP content and antioxidant activity of wounded carrots. 2. Materials and methods 2.1. Chemical reagent Methanol was purchased from Guangdong Guanghua Sci-Tech CO., Ltd. (Guangdong, China). Acetone was purchased from Shanghai Lingfeng Chemical Regent CO., Ltd. (Shanghai, China). Diphenyliodonium iodide (DPI) was purchased from Tokyo Chemical Industry CO., Ltd. (Tokyo, JAPAN). Nitrotetrazolium blue chloride (NBT) and L-Phenylalanine were purchased from Shanghai Ryon Biological Technology CO., Ltd. (Shanghai, China). Folin– Ciocalteu reagent, ascorbic acid and glucose oxidase were purchased from Beijing Solarbio Science & Technology CO., Ltd. (Beijing, China). Glucose, hydroxyammonium chloride, p-aminophenylsulfonic acid, and a-naphthylamine were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used in this study were obtained from Sigma– Aldrich (St. Louis, MO, USA). All chemicals were of reagent grade. 2.2. Plant material, treatment and storage Fresh carrots (Daucus carota L. cv. Sanhongliucun) were purchased from a local wholesale market in Nanjing, P.R. China. After transferred to the laboratory, all carrots were washed and selected for uniform size (20–24 cm in length, 2.5–3 cm in diameter of equator), color, firmness, shape and free from blemishes. The selected carrots were conditioned at 10 °C overnight prior to two independent experiments. In the first experiment, wounding treatment was carried out according to the method of Surjadinata and Cisneros-Zevallos (2012). Carrots from each group were cut into slices, pies and shreds to create different wounding intensities. The wounding intensity of each cut type was defined by the ratio of the new surface area (A) created by wounding in cm2 over the tissue weight (W) in g. The calculated wounding intensities (A/W) were 4.9, 6.4 and 18.5 cm2/g, respectively, for slices, pies, and shreds. Whole carrots with 0.00 cm2/g of A/W were used as controls. After
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wounding treatment, slices, pies, and shreds were placed into 15 cm 10 cm 4 cm rigid polypropylene containers and stored at 4, 10 and 20 °C with 85–90% relative humidity for 7, 4 and 2 days, respectively. During storage, samples were taken and frozen in liquid nitrogen, and stored at 80 °C until use. In the second experiment, the impact of ROS on wound-induced accumulation of phenolic compounds in carrots was conducted. Shredded carrots were selected and immersed in the following solutions for 3 min: water, 300 lM diphenyliodonium iodide (DPI, ROS inhibitor), 50 lM glucose, 50 lM glucose/glucose oxidase (G/GO, ROS elicitor). Each treatment was applied to three replications. The concentrations of the chemicals were chosen based on the literatures (Jacobo-Velázquez et al., 2011; OrozcoCárdenas, Narváez-Vásquez, & Ryan, 2001) and our preliminary experiments. After treatment, all samples were then air-dried for approximately 15 min and stored at 20 °C with 85–90% RH for 48 h. During storage, samples were taken every 12 h, frozen in liquid nitrogen and stored at 80 °C until use. 2.3. Measurements of O 2 and H2O2 O 2 production was measured following the method of Elstner (1976) with some modifications. Five grams of fresh tissue was ground in 20 mL of 50 mM phosphate buffer (pH 7.8). The homogenate was centrifuged at 10,000g for 20 min at 4 °C. A sample of the crude extract (1 mL) was added to 1 mL of 1 M hydroxyammonium chloride and incubated at 25 °C for 1 h. Then 2 mL ether was added to the incubation mixture in order to prevent the interference of pigment and the mixture was centrifuged at 10,000g for 5 min. After that 1 mL mixture from the water layer, 1 mL of 17 mM p-aminophenylsulfonic acid and 7 mM a-naphthylamine (dissolved in glacial acetic acid: H2O = 3:1) were added and the mixture incubated at 25 °C for a further 20 min, the absorbance was measured at 530 nm. O 2 production was calculated against the standard curve using sodium nitrite as a standard and expressed as nM [NO2] g1 min1. For analysis of H2O2 content, three grams of fresh tissue was ground in 10 mL of chilled acetone. The homogenate was centrifuged at 10,000g for 20 min at 4 °C. The supernatant was collected immediately for H2O2 analysis using the method of Patterson, Mackae, and Ferguson (1984). The content of H2O2 was expressed as lM g1. 2.4. Total soluble phenolics (TSP) content assay TSP content was assayed following the method of Slinkard and Singleton (1977) with some modifications. Frozen tissue samples (5 g) were extracted using a mortar and pestle with 25 mL of cold methanol. The homogenate was centrifuged at 13,000g for 15 min at 4 °C and the supernatant was used for the TSP content assay. A sample of the crude extract (500 lL) was added to 1.5 mL of distilled water and 1 mL of FolinCiocalteu reagent, after that 1 mL 7.5% (w/v) Na2CO3 solution was added. The mixtures were incubated at 25 °C for 2 h before reading at 765 nm. The TSP content was expressed as milligrams of gallic acid (GAE) per kilogram. 2.5. Measurement of antioxidant capacity Antioxidant capacity was measured using 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical-scavenging activity as reported by De Ancos, Sgroppo, Plaza, and Cano (2002) with some modifications. The same extracts prepared for TSP assay were used for determination of antioxidant capacity. An aliquot of 0.2 mL of the TSP extract was added to 2.8 mL of 0.12 mM DPPH solution (prepared with ethanol). After incubated at 25 °C for 30 min, the absorbance was measured at 525 nm, and 0.2 mL of 80% ethanol
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adding to 2.8 mL of DPPH reagent was used as the control solution. The results were expressed as % DPPH inhibition and calculated with the following equation:
% DPPH inhibition ¼ ½ðA0 A1 Þ=A0 100 with A0, the absorbance of the control; and A1, the absorbance of the sample. 2.6. Phenylpropanoid metabolism-related enzymes assays Phenylalanine ammonium lyase (PAL) activity was assayed following the method of Assis, Maldonado, Muñoz, Escribano, and Merodio (2001). Four grams of fresh tissue was homogenized with 20 mL of ice-cold 50 mM sodium borate buffer (pH 8.8, containing 5 mM b-mercaptoethanol, 2 mM EDTA and 40 g/L PVP) and ground at 4 °C. The homogenate was centrifuged at 13,000g for 20 min at 4 °C and the supernatant was used for the enzyme assay. One milliliter of crude enzyme extraction solution was incubated with 2 mL of sodium borate buffer (50 mM, pH 8.8, containing 5 M bmercaptoethanol and 2 mM EDTA) and 0.5 mL of L-phenylalanine (20 mM), for 60 min, at 37 °C. The reaction was stopped by adding 0.1 mL of 6 M HCl. One unit of PAL activity was equal to a change of 0.01 at 290 nm per min, and expressed as U mg1 protein. Cinnamate-4-hydroxylase (C4H) activity was determined according to the method of Lamb and Rubery (1975) with some modifications. Four grams of fresh tissue was homogenized with 20 mL of ice-cold 50 mM Tris-HCl buffer (pH 8.9; containing 15 mM b-mercaptoethanol, 4 mM MgCl2, 5 mM ascorbic acid, 10 lM Leupeptin, 1 mM PMSF, 0.15% w/v PVP and 10% glycerol) and ground at 4 °C. The homogenate was centrifuged at 13,000g for 20 min at 4 °C and the supernatant was used for the enzyme assay. The reaction mixture contained 2.5 mL of 50 mM Tris-HCl buffer (pH 8.9; containing 2 lM trans-cinnamic acid, 2 lM NADPNa2, 5 lM G-6-PNa2) and 0.5 mL crude enzyme. The mixture was blended and incubated at 25 °C for 30 min and the reaction was stopped by adding 100 lL of 6 M HCl. The absorbance was measured at 340 nm before and after incubation. One unit of C4H activity was equal to a change of 0.01 in absorbance per min, and expressed as U mg1 protein. 4-coumarate coenzyme A ligase (4CL) activity was assayed as described by Knobloch and Hahlbrock (1975) with some modifications. Four grams of fresh tissue was homogenized with 20 mL of ice-cold 50 mM Tris-HCl buffer (pH 8.9; containing 15 mM bmercaptoethanol, 4 mM MgCl2, 5 mM ascorbic acid, 10 lM leupeptin, 1 mM PMSF, 0.15% w/v PVP and 10% glycerol) and ground at 4 °C. The reaction mixture contained 2 mL of 5 mM MgCl2, 0.5 mL of 5 mM adenosine triphosphate (ATP), 0.05 mL of 0.4 mM coenzyme A (CoA), 0.05 mL of 0.6 mM p-cumaric acid and 0.5 mL crude enzyme. The mixture was blended and incubated at 40 °C for 10 min and the reaction was stopped by adding 100 lL of 6 M HCl. The absorbance was measured at 333 nm before and after incubation. One unit of 4CL activity was equal to a change of 0.01 in absorbance per min, and expressed as U mg1 protein. 2.7. Antioxidant enzymes assays Five grams of fresh tissue were homogenized with 25 mL of icecold extraction buffers containing 0.2 g of PVPP and ground at 4 °C. For superoxide dismutase (SOD), the extraction buffer was 100 mM sodium phosphate (pH 7.8). For catalase (CAT) and ascorbate peroxidase (APX), the extraction buffer was 100 mM sodium phosphate (pH 7.0) while the buffer of APX containing 0.1 mM EDTA, 1 mM ascorbic acid and 1% polyvinyl-pyrrolidone. SOD activity was determined by the method of Rao, Paliyath, and Ormrod (1996). SOD was assayed by mixing 1.75 mL of 50 mM phosphate buffer (pH 7.8), 0.3 mL of 130 mM methionine,
0.3 mL of 750 lM NBT, 0.3 mL of 20 lM riboflavin, 0.3 mL of 100 lM EDTA-Na2, and 0.05 mL enzyme extract. Riboflavin was added at the end, and the reaction mixture was shaken and immediately placed under 4000 lx fluorescent lamp. One unit of SOD activity was defined as a change of 1 per min in OD560 and results were expressed as U mg1 protein. CAT activity was analyzed based on Wang and Tian (2005) with certain modifications. The reaction mixture consisted of 1.4 mL sodium phosphate buffer (50 mM, pH 7.0), 1 mL H2O2 (40 mM) and 0.6 mL enzyme extract. H2O2 was added at the end. One unit of CAT activity was defined as a change of 0.01 per min in OD240 and results were expressed as U mg1 protein. APX activity was determined by the method of Nakano and Asada (1989). The reaction mixture consisted of 2 mL sodium phosphate buffer (50 mM, pH 7.0), 0.8 mL H2O2 (2 mM) and 0.2 mL enzyme extract. H2O2 was added at the end. One unit of APX was defined a change of 0.01 per min in OD290, and results were expressed as U mg1 protein. Protein content in crude enzyme extracts was measured according to Bradford (1976) using bovine serum albumin as a standard. 2.8. Statistical analysis Experiments were performed using a completely randomized design and each treatment was replicated three times. All data were expressed as the mean ± standard error (SE) of three replicates and subjected to statistical analysis with SPSS 13.0 (SPSS Inc., Chicago, Illinois, US). The data were analyzed by one-way analysis of variance (ANOVA). Mean separations were performed using Duncan’s multiple range test, and differences at p < 0.05 were considered to be significant. In each figure, data points carrying different letters for the same storage time indicate statistically significant differences. 3. Results 3.1. Effects of wounding intensity and storage temperature on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity of wounded carrots As shown in Table 1, the O 2 production in all three cutting styles of carrots changed slightly and remained at relatively low levels at 4 °C. However, a greater accumulation of H2O2 content was observed in shreds compared with slices and pies. Increased wounding intensity and higher storage temperature both increased the ROS production of wounded carrots. The average values of O 2 and H2O2 in shreds were much higher than those of other two cutting styles during storage at 10 °C and 20 °C. The changes of TSP content and DPPH radical scavenging activity in all samples increased continuously during storage. Increased wounding intensity and higher storage temperature both accelerated the increase of TSP content and DPPH radical scavenging activity in wounded carrots. At the end of storage, the maximum value of TSP content was obtained in shreds at 20 °C, which showed a 4.8-fold increase compared to day 0 (233.13 mg GAE/kg for control). Correspondingly, the highest value of DPPH radical scavenging activity was observed in shredded carrots at 20 °C, which was 6.5 times higher than its initial value. 3.2. Effects of DPI and G/GO treatments on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity of shredded carrots Shredded carrots stored at 20 °C were selected for the second experiment. As shown in Table 2, the O 2 production in H2O treated samples decreased during the first 12 h and then experienced a
Table 1 a Effects of wounding intensity and storage temperature on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity in wounded carrots. Storage time (days)
1 O min1) 2 production (nM [NO2] g
H2O2content (lM g1)
Slices
Pies
Shreds
Slices
Pies
Shreds
Slices
Pies
Shreds
Slices
Pies
Shreds
4 °C
0 1 3 5 7
1.34 ± 0.04abA 1.26 ± 0.03cB 1.38 ± 0.03aA 1.31 ± 0.04bcB 1.31 ± 0.01bcA
1.36 ± 0.03bA 1.45 ± 0.04aA 1.38 ± 0.01bA 1.44 ± 0.04aA 1.33 ± 0.03bA
1.40 ± 0.03abA 1.44 ± 0.06aA 1.21 ± 0.03 dB 1.36 ± 0.05bcAB 1.30 ± 0.04bcA
8.59 ± 0.07bA 8.13 ± 0.14cC 8.97 ± 0.06aB 7.61 ± 0.08dC 8.51 ± 0.03bB
8.56 ± 0.12bA 9.33 ± 0.06aB 8.71 ± 0.10bC 9.27 ± 0.11aB 7.50 ± 0.15cC
8.61 ± 0.05eA 10.25 ± 0.13dA 14.20 ± 0.09cA 15.05 ± 0.12aA 14.44 ± 0.16bA
234.42 ± 4.12cA 278.31 ± 6.20bA 284.28 ± 6.05bB 315.15 ± 7.57aB 302.55 ± 10.62aB
235.26 ± 5.41cA 266.16 ± 5.66bA 298.17 ± 7.22aB 300.81 ± 12.10aB 310.05 ± 13.84aB
233.13 ± 6.83dA 244.83 ± 10.35 dB 334.53 ± 8.96cA 360.84 ± 4.83bA 445.98 ± 9.33aA
12.16 ± 0.46dA 15.44 ± 1.20cA 15.86 ± 0.64bcA 19.75 ± 1.01aA 17.72 ± 1.52bB
12.27 ± 0.65dA 14.60 ± 1.04cA 16.30 ± 0.26bA 17.37 ± 0.92abB 18.66 ± 1.28aB
12.12 ± 0.82dA 13.43 ± 1.45dA 16.70 ± 0.71cA 20.13 ± 0.50bA 28.50 ± 0.88aA
10 °C
0 1 2 3 4
1.34 ± 0.04dA 1.59 ± 0.04cB 1.56 ± 0.05cB 1.91 ± 0.08aB 1.73 ± 0.05bC
1.36 ± 0.03cA 1.86 ± 0.03bA 1.88 ± 0.04abA 1.92 ± 0.11abB 1.99 ± 0.06aB
1.40 ± 0.03dA 1.47 ± 0.07cdC 1.59 ± 0.06cB 2.32 ± 0.09bA 2.70 ± 0.10aA
8.59 ± 0.07dA 9.08 ± 0.04cB 11.26 ± 0.09bB 9.57 ± 0.07cC 13.70 ± 0.13aB
8.56 ± 0.12eA 8.79 ± 0.06dC 9.27 ± 0.08cC 10.68 ± 0.10aB 9.94 ± 0.06bC
8.61 ± 0.05eA 10.28 ± 0.07dA 12.56 ± 0.05cA 18.77 ± 0.14bA 19.23 ± 0.18aA
234.42 ± 4.12dA 342.09 ± 16.59cA 457.68 ± 18.51bA 462.72 ± 9.92bB 493.74 ± 11.46aB
235.26 ± 5.41eA 341.40 ± 7.43dA 367.59 ± 5.14cB 419.37 ± 16.09bC 460.38 ± 23.30aB
233.13 ± 6.83eA 263.85 ± 12.06 dB 387.57 ± 13.73cB 535.35 ± 25.82bA 669.84 ± 14.71aA
12.16 ± 0.46dA 17.85 ± 1.43cA 30.13 ± 0.66bA 30.44 ± 1.81bB 34.50 ± 0.94aB
12.27 ± 0.65eA 17.28 ± 0.92dA 22.12 ± 1.40cB 26.18 ± 1.30bC 29.39 ± 1.96aC
12.12 ± 0.82dA 14.96 ± 0.65 dB 22.67 ± 3.22cB 37.61 ± 2.41bA 44.84 ± 2.77aA
20 °C
0 0.5 1 1.5 2
1.34 ± 0.04bA 1.32 ± 0.05bB 1.26 ± 0.04bB 1.04 ± 0.05cC 1.65 ± 0.08aB
1.36 ± 0.03cA 1.44 ± 0.04cAB 2.05 ± 0.05aA 1.62 ± 0.08bB 1.34 ± 0.09cC
1.40 ± 0.03dA 1.53 ± 0.10dA 1.88 ± 0.14cA 3.48 ± 0.11aA 2.64 ± 0.15bA
8.59 ± 0.07cA 7.37 ± 0.06dC 7.46 ± 0.11dC 9.68 ± 0.08bC 12.01 ± 0.07aC
8.56 ± 0.12eA 9.72 ± 0.07 dB 11.13 ± 0.10cB 13.53 ± 0.02bB 22.84 ± 0.11aB
8.61 ± 0.05eA 11.19 ± 0.12dA 17.55 ± 0.05cA 26.12 ± 0.13bA 33.44 ± 0.17aA
234.42 ± 4.12dA 273.36 ± 5.38cAB 286.95 ± 11.42cC 364.44 ± 22.01bC 390.42 ± 11.17aC
235.26 ± 5.41eA 284.67 ± 4.45dA 335.76 ± 7.93cB 425.43 ± 18.22bB 609.42 ± 15.56aB
233.13 ± 6.83eA 265.17 ± 9.02 dB 436.23 ± 20.70cA 797.43 ± 13.64bA 1128.72 ± 16.55aA
12.16 ± 0.46cA 15.17 ± 1.38bA 16.33 ± 1.52bC 21.68 ± 0.85aC 24.42 ± 1.84aC
12.27 ± 0.65eA 15.64 ± 1.82dA 19.85 ± 0.87cB 27.13 ± 1.26bB 40.30 ± 2.55aB
12.12 ± 0.82dA 14.08 ± 2.47dA 29.21 ± 1.64cA 53.72 ± 3.68bA 78.27 ± 2.40aA
TSP content (mg GAE/kg)
DPPH radical scavenging activity (%)
a Data are expressed as the mean ± SD (n = 3). Values in the same storage temperature with different letters were significantly different at p < 0.05. Lowercase letters represented significant difference among storage time factors, and capital letters represented significant difference among cutting style factors.
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Storage temperature
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Table 2 a Effects of DPI and G/GO treatments on O 2 production, H2O2 content, TSP content and DPPH radical scavenging activity in shredded carrots during 2 days storage at 20 °C. Storage time (hours) 0 12 24 36 48
Shreds-H2O 1.06 ± 0.04bcB 0.85 ± 0.02 dB 0.99 ± 0.03cA 1.08 ± 0.06bA 1.29 ± 0.05aA
Shreds-DPI 0.94 ± 0.02aC 0.62 ± 0.03dC 0.54 ± 0.01eB 0.72 ± 0.05cB 0.83 ± 0.04bC
Shreds-glucose 1.08 ± 0.03bAB 0.88 ± 0.01cB 1.02 ± 0.05bA 1.08 ± 0.02bA 1.32 ± 0.04aA
Shreds-G/GO 1.13 ± 0.04abA 1.05 ± 0.03cdA 1.00 ± 0.03dA 1.10 ± 0.02bcA 1.19 ± 0.06aB
H2O2 content (lM g1)
0 12 24 36 48
6.84 ± 0.06eA 7.53 ± 0.03 dB 8.24 ± 0.05cC 9.65 ± 0.11bA 10.26 ± 0.08aA
6.16 ± 0.05 dB 6.63 ± 0.12cC 7.02 ± 0.02aD 7.11 ± 0.06aC 6.88 ± 0.07bD
6.88 ± 0.04eA 7.60 ± 0.07 dB 8.35 ± 0.09cB 9.23 ± 0.05bB 9.97 ± 0.09aB
6.85 ± 0.07eA 10.13 ± 0.10aA 8.98 ± 0.04dA 9.27 ± 0.08cB 9.47 ± 0.03bC
TSP content (mg GAE/kg)
0 12 24 36 48
176.16 ± 3.42eA 210.37 ± 2.53dA 301.65 ± 5.05cB 354.50 ± 14.55bB 412.35 ± 16.86aB
175.92 ± 4.84dA 184.29 ± 5.3 dB 204.45 ± 6.90cC 227.94 ± 11.43bC 261.63 ± 8.23aC
177.55 ± 3.68eA 211.51 ± 6.56dA 303.96 ± 12.45cB 361.92 ± 10.33bB 418.14 ± 14.72aB
176.34 ± 2.01eA 219.23 ± 5.87dA 332.49 ± 10.6cA 417.48 ± 15.16bA 466.38 ± 16.21aA
DPPH radical scavenging activity (%)
0 12 24 36 48
9.31 ± 0.72eA 14.48 ± 0.83dA 18.30 ± 1.2cB 23.83 ± 1.70bB 27.56 ± 1.39aB
9.22 ± 0.56cA 10.12 ± 1.73cB 11.68 ± 1.63bcC 13.61 ± 1.52bC 16.54 ± 1.26aC
9.26 ± 0.36eA 14.73 ± 1.47dA 18.77 ± 1.44cB 24.02 ± 0.95bB 28.02 ± 0.56aB
9.64 ± 1.13eA 14.61 ± 0.65dA 21.48 ± 0.85cA 27.99 ± 1.24bA 31.45 ± 1.08aA
1 O min1) 2 production (nM [NO2] g
a Data are expressed as the mean ± SD (n = 3). Values with different letters were significantly different at p < 0.05. Lowercase letters represented significant difference among storage time factors, and capital letters represented significant difference among treatment factors.
gradual increase in subsequent storage period. DPI treatment markedly inhibited the production of O 2 , and the significant (p < 0.05) difference was observed throughout the whole storage period. Similarly, the lower level of H2O2 was also observed in DPI treated samples. In contrast, the use of G/GO improved the initial production of O 2 , and brought a 47.9% increase of H2O2 content at the 12 h compared to 0 h. The TSP content and DPPH radical scavenging activity increased gradually during storage, whereas, their initial value or rising trend was weakened compared with un-immersed shreds. DPI treatment significantly (p < 0.05) restrained the increase of TSP content and DPPH radical scavenging activity in shredded carrot. In contrast, G/GO treatment induced the increase of TSP content and DPPH radical scavenging activity, and the values of these two parameters were 15.4% and 16.5% higher than control at 36 h, respectively. 3.3. Effects of DPI and G/GO treatments on PAL, C4H and 4CL activities of shredded carrots As shown in Fig. 1A, PAL activity in control shreds had a great increase during the first 24 h, thereafter kept at a high level in the second day. Similar as the pattern of PAL activity, the activities of C4H and 4CL in control samples increased gradually during the first 36 h (Fig. 1B and C). DPI treatment significantly (p < 0.05) inhibited the increase the activities of PAL, C4H and 4CL during the entire storage time. In contrast, G/GO treatment enhanced the activity of PAL and the significant (p < 0.05) difference was observed at the 24 h compared to control samples. The C4H activity of G/GO treated shreds experienced a pronounced increase from the 12 h to 36 h during storage, while for 4CL, the obvious increase was observed between 24 h and 36 h. 3.4. Effects of DPI and G/GO treatments on SOD, CAT and APX activities of shredded carrots SOD activity in control samples decreased markedly during the first 12 h, and then experienced a gradual increase afterward (Fig. 2A). DPI treatment significantly (p < 0.05) inhibited the increase of SOD activity during the whole storage time. Conversely, G/GO treatment enhanced the activity of SOD at 12 h, while no
significant difference (p > 0.05) was observed in the following storage time. As shown in Fig. 2B, the CAT activity in control shreds increased steadily during 48 h of storage. However, no significant changes were observed in DPI treated samples. The use of G/GO could effectively induce the activity of CAT apart from the last 12 h of storage. Unlike with the change of CAT, the APX activity in control shreds increased at 12 h, then decreased slightly at 24 h, thereafter increased dramatically (Fig. 2C). G/GO treatment improved the APX activity during the first 24 h, however, a lower value was observed at later storage period. Although the APX activity in DPI treated samples was relatively lower than control samples, an obvious increase was also observed between 12 h and 24 h. 4. Discussion ROS are generated in plant tissue during plant growth and development and have been recognized as signal molecules in plant defense responses (Vranová, Inz´e, & Van Breusegem, 2002). Many postharvest abiotic stresses such as temperature, atmospheric composition, wounding and phytohormones can induce ROS generation which affects physiological, metabolic properties and quality of fruits and vegetables (Hodges, Lester, Munro, & Toivonen, 2004). The physiological role of ROS on the accumulation of phenolic antioxidants in wounded carrots was previously reported (Jacobo-Velázquez et al., 2011, 2015). This study has gone further to understand the effects of storage temperature on ROS levels and TSP content in wounded carrots. As shown in Table 1, increased wounding intensity enhanced the formation of O 2 , the generation of H2O2 and induced a higher accumulation of phenolics in shredded carrots. This result confirmed the previous reports that the biosynthesis of phenolics in wounded carrot increased with increasing wounding intensity (Du et al., 2012; Surjadinata & Cisneros-Zevallos, 2012) and that the wound-induced accumulation of phenolic compounds in carrots was associated with the increase of ROS levels (JacoboVelázquez et al., 2011). Moreover, we found that higher storage temperature promoted the production of ROS and caused more TSP content in wounded carrots, suggesting that the application of higher storage temperature may increase wound-induced phenolic accumulation through enhancing ROS levels. In plant cells,
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Fig. 1. Effects of DPI and G/GO treatments on activities of PAL (A), C4H (B), and 4CL (C) in shredded carrots during 2 days storage at 20 °C. Data are expressed as the mean ± SD (n = 3). Data points carrying different letters for the same storage time indicate statistically significant differences (p < 0.05).
Fig. 2. Effects of DPI and G/GO treatments on activities of SOD (A), CAT (B), and APX (C) in shredded carrots during 2 days storage at 20 °C. Data are expressed as the mean ± SD (n = 3). Data points carrying different letters for the same storage time indicate statistically significant differences (p < 0.05).
the mitochondrial electron transport chain is a major site of ROS production (Møller, 2001). Higher storage temperature may provoke the mitochondrial respiratory activity, thereby inducing the production of ROS and the biosynthesis of phenolic compounds in wounded carrots. As an important category of phytochemicals,
phenolic compounds have been documented the main contributor to the antioxidant capacity of plants (Scalbert, Johnson, & Saltmarsh, 2005). The observed increase of antioxidant capacity (Table 1) in wounded carrots can be attributed to their higher TSP content.
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In the second experiment, to confirm the vital role of ROS on wound-induced phenolics accumulation, shredded carrots were used to assay the impact of ROS inhibitor or elicitor on changes of TSP content. Our results showed that treatment with the NADPH oxidase inhibitor–DPI could significantly reduce the generation of O 2 and H2O2 and suppressed and the accumulation of phenolics in shredded carrots and (Table 2). This result was consistent with the previous study of Jacobo-Velázquez et al. (2011), who found that the accumulation of phenolic compounds was almost completely blocked in DPI-treated carrot shreds. Glucose oxidase (GO) is a flavoprotein which catalyses the oxidation of b-Dglucose to produce D-glucono-d-lactone and H2O2 (Bankar, Bule, Singhal, & Ananthanarayan, 2009). In the present study, treatment with G/GO significantly enhanced H2O2 content of shredded carrots and markedly increased TSP content after 24 h of storage (Table 2). These observations confirmed previous reports of Jacobo-Velázquez et al. (2011, 2015) that ROS played a key role as signaling molecules for the wound-induced accumulation of phenolic compounds in wounded carrots. PAL, C4H and 4CL are three pivotal enzymes that catalyse the synthesis of phenolic compounds in phenylpropanoid pathways in plants (Weisshaar & Jenkins, 1998). PAL catalyses the conversion of phenylalanine to trans-cinnamic acid, and then hydroxylated to p-coumaric acid in the presence of C4H. As the metabolism of phenolic compounds continues, p-coumaric is transformed to p-coumaroyl CoA by 4CL and finally formed to caffeoylquinic acids and its derivatives, which are the major increased phenolic profiles in fresh-cut carrot (Becerra-Moreno et al., 2012; Jacobo-Velázquez et al., 2015). Many studies have demonstrated that increases of PAL, C4H and 4CL activities would result in the accumulation of phenolic compounds (Chen et al., 2006; Liu et al., 2014). Our experiment found that the activity of PAL was inhibited by DPI treatment, while activated by G/GO treatment (Fig. 1A). Likewise, the activities of C4H and 4CL showed similar tendency with PAL activity and matched with the accumulation of TSP content (Fig. 1B and C). These results were consistent with the findings of Jacobo-Velázquez et al. (2015) on the expression of genes encoding those enzymes, suggesting that ROS can affect phenolics biosynthesis in wounded carrots by inducing phenylpropanoid pathway enzymes. Also, it was noteworthy that the ‘‘oxidative burst” preceded the peak of these three enzymes, which may indicate that there exists a signal transduction time for carrot cell to mobilize these phenylpropanoid pathway enzymes. In order to maintain the concentration of ROS at relatively low level, plants have evolved mechanisms to scavenge these toxic and reactive substances by antioxidant compounds (e.g., phenolic compounds) and by antioxidant enzymes such as SOD, CAT and APX. SOD, as the major O 2 scavenging enzyme, catalyses the disproportionation of O 2 radicals into H2O2 and O2, while both CAT and APX catalyze the degradation of H2O2 to H2O and O2. In the present work, the activities of SOD, CAT and APX were suppressed in DPI-treated shreds (Fig. 2) and matched with the lower levels of O 2 and H2O2. This result confirmed the previous work by Jacobo-Velázquez et al. (2011) on those same enzymes. As for G/GO-treated shreds, the activities of SOD, CAT and APX were significantly enhanced, however, the contents of ROS were maintained at high level. This result could be caused by the G/GO reaction that produces H2O2, which in turn induce antioxidant enzymes activity. Previous studies have also shown that pretreatment with H2O2 enhanced antioxidant enzymes activity, thus inducing protection from chilling-induced oxidative stress in maize and tobacco (Gechev et al., 2002; Prasad, Anderson, Martin, & Stewart, 1994). These results confirmed previous work of Jacobo-Velázquez et al. (2011, 2015) that antioxidant enzymes played important role on modulating ROS levels in wounded carrots.
5. Conclusions This study confirmed previous findings that increased wounding intensity induced the formation of O 2 , the generation of H2O2, and the accumulation of phenolic compounds in wounded carrots. Moreover, higher storage temperature strengthened ROS production and phenolics accumulation in wounded carrots through enhancing ROS levels. ROS inhibitor DPI inhibited ROS generation, suppressed the activities of PAL, C4H, 4CL and caused a lower accumulation of phenolic compounds in shredded carrots confirming previous reports. Whereas, ROS elicitor G/GO enhanced ROS generation, induced the activities of these enzymes and brought a higher accumulation of phenolics. Thus, our results confirmed that ROS are essential for mediating wound-induced phenolics accumulation in carrots and suggested that increase temperature enhanced the accumulation of phenolics. Acknowledgments This study was financially supported by the National Natural Science Foundation of China (No. 31471632). References Alegria, C., Pinheiro, J., Duthoit, M., Gonçalves, E. M., Moldão-Martins, M., & Abreu, M. (2012). Fresh-cut carrot (cv. Nantes) quality as affected by abiotic stress (heat shock and UV-C irradiation) pre-treatments. LWT – Food Science and Technology, 48(2), 197–203. Artés-Hernández, F., Rivera-Cabrera, F., & Kader, A. A. (2007). Quality retention and potential shelf-life of fresh-cut lemons as affected by cut type and temperature. Postharvest Biology Technology, 43(2), 245–254. Assis, J. S., Maldonado, R., Muñoz, T., Escribano, M. I., & Merodio, C. (2001). Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biology and Technology, 23(1), 33–39. Babic, I., Amiot, M. J., Nguyen-The, C., & Aubert, S. (1993). Changes in phenolic contents in fresh ready-to-use shredded carrots during storage. Journal of Food Science, 58(2), 351–356. Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase – an overview. Biotechnology Advances, 27(4), 489–501. Becerra-Moreno, A., Benavides, J., Cisneros-Zevallos, L., & Jacobo-Velázquez, D. A. (2012). Plants as biofactories: glyphosate-induced production of shikimic acid and phenolic antioxidants in wounded carrot tissue. Journal of Agricultural and Food Chemistry, 60(45), 11378–11386. Becerra-Moreno, A., Redondo-Gil, M., Benavides, J., Nair, V., Cisneros-Zevallos, L., & Jacobo-Velázquez, D. A. (2015). Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot. Frontiers in Plant Science, 6, 837. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72(1–2), 248–254. Breusegem, F. V., Vranová, E., Dat, J. F., & Inzé, D. (2001). The role of active oxygen species in plant signal transduction. Plant Science, 161(3), 405–414. Campos-Vargas, R., & Saltveit, M. E. (2002). Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce. Plant Physiology, 114(1), 73–84. Chen, J. Y., Wen, P. F., Kong, W. F., Pan, Q. H., Wan, S. B., & Huang, W. D. (2006). Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development. Journal of Plant Physiology, 163(2), 115–127. De Ancos, B., Sgroppo, S., Plaza, L., & Cano, M. P. (2002). Possible nutritional and health related value promotion in orange juice preserved by high-pressure treatment. Journal of Agricultural and Food Chemistry, 82(8), 790–796. Du, W. X., Avena-Bustillos, R. J., Breksa, A. P., III, & McHugh, T. H. (2012). Effect of UV-B light and different cutting styles on antioxidant enhancement of commercial fresh-cut carrot products. Food Chemistry, 134(4), 1862–1869. Elstner, E. F. (1976). Inhibition of nitrite formation hydroxylam–mouium–chloride: a simple assay for superoxide dismutase. Analytical Biochemistry, 70, 616–620. Gechev, T. S., Gadjev, I., Van Breusegem, F., Inzé, D., Dukiandjiev, S., Toneva, V., & Minkov, I. (2002). Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. Cellular and Molecular Life Sciences CMLS, 59(4), 708–714. Heredia, J. B., & Cisneros-Zevallos, L. (2009). The effect of exogenous ethylene and methyl jasmonate on pal activity, phenolic profiles and antioxidant capacity of carrots (Daucus carota) under different wounding intensities. Postharvest Biology Technology, 51(2), 242–249. Hodges, D. M., & Toivonen, P. M. A. (2008). Quality of fresh-cut fruits and vegetables as affected by exposure to abiotic stress. Postharvest Biology Technology, 48(2), 155–162.
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