Article pubs.acs.org/JAFC
Blue Light Irradiation Affects Anthocyanin Content and Enzyme Activities Involved in Postharvest Strawberry Fruit Feng Xu,† Shifeng Cao,‡ Liyu Shi,§ Wei Chen,§ Xinguo Su,# and Zhenfeng Yang*,§ †
School of Marine Sciences, Ningbo University, Ningbo 315211, People’s Republic of China Nanjing Research Institute for Agricultural Mechanization, Ministry of Agriculture, Nanjing 210014, People’s Republic of China § College of Biological and Environmental Sciences, Key Laboratory of Fruits and Vegetables Postharvest and Processing Technology Research of Zhejiang Province, Zhejiang Wanli University, Ningbo 315100, People’s Republic of China # Guangdong Food and Drug Vocational College, Guangzhou 510520, People’s Republic of China ‡
ABSTRACT: Blue light irradiation was applied to postharvest strawberry fruit to explore its influence on anthocyanin content and anthocyanin biosynthetic enzyme activities. Strawberry fruit was irradiated with blue light at 40 μmol m−2 s−1 for 12 days at 5 °C. The results indicated that blue light treatment improved total anthocyanin content in strawberry fruit during storage. Meanwhile, the treatment increased the activities of glucose-6-phosphate, shikimate dehydrogenase, tyrosine ammonia-lyase, phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, 4-coumarate/coenzyme A ligase, dihydroflavonol-4-reductase, chalcone synthase, flavanone-3-β-hydroxylase, anthocyanin synthase, and UDP-glycose flavonoid-3-O-glycosyltranferase, which suggested that the enhancement of anthocyanin concentration by blue light might result from the activation of its related enzymes. Blue light might be proposed as a supplemental light source in the storage of strawberry fruit to improve its anthocyanin content. KEYWORDS: strawberry fruit, blue light, anthocyanin, postharvest
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INTRODUCTION Anthocyanins are red, orange, purple, or blue water-soluble pigments occurring in fruits and vegetables, which play important biological roles in protecting plants against various biotic and abiotic stresses and in furnishing flowers and fruits with distinct colors to attract insects and animals for pollination and seed dispersal.1 In addition to colorant properties, interest in anthocyanins has intensified due to their reported role in reducing the risk of coronary heart disease, cancer, and stroke.2 Therefore, consumption of fruit rich in anthocyanins may be beneficial to human health.3 It is vital to understand the regulation of anthocyanin biosynthesis thoroughly to develop anthocyanin-rich foods, thereby meeting the increasing demand for health-promoting compounds in our diet. To date, the biosynthesis pathway of anthocyanins and flavonoids has been almost completely elucidated as shown in Figure 1, and most of the structural genes encoding the enzymes responsible for each step have been isolated from different sources.4,5 Light is one of the most important environmental factors for plants, as a source of energy and in many other respects including anthocyanin synthesis.6 Various types of lights might have distinct effects.7−9 Among the different light wavelengths, blue light is one of the most effective in enhancing anthocyanin biosynthesis.10,11 For example, anthocyanin production in apples had a strong dependence on both intensity and quality of light. Blue-violet and UV light were most effective, and far red was least effective, or even inhibitory.7 Illumination with blue (both 455 and 470 nm) light-emitting diodes (LEDs) light resulted in higher anthocyanins content in red lettuce.12 It is also reported that anthocyanin accumulation showed higher induction levels under blue light (465 nm) relative to green and red light during fruit development in strawberries.14 © 2014 American Chemical Society
Figure 1. Simplified pathway for anthocyanin and flavonoid biosynthesis. G6PDH, glucose-6-phosphate; SKDH, shikimate dehydrogenase; PAL, phenylalanine ammonia-lyase; TAL, tyrosine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose:flavonoid glucosyltransferase.
Strawberry is one of the most commonly consumed berries and a good source of anthocyanins, which have shown potent antioxidant properties against chemically generated radicals.13 Received: Revised: Accepted: Published: 4778
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PAL and TAL assays were conducted according to the methods of Zucker and Kozukue et al.,22,23 respectively. One unit of PAL activity was expressed as the amount of enzyme causing an increase in A290 of 0.001 unit h−1. One unit of TAL was defined as the amount of enzyme causing an increase in A333 of 0.01 unit h−1. According to the method of Lamb et al.,24 C4H activity was assayed in 50 mM phosphate buffer containing 2 mM 2-mercaptoethanol, 0.5 mM NADPH, and 2 mM trans-cinnamic acid, in a final volume of 2 mL of reaction buffer. The mixture was incubated for 1 h at 37 °C. The reaction was terminated by adding 6 M HCl, and 1 unit of C4H activity was expressed as the amount of enzyme causing an increase in A290 of 0.001 unit h−1. 4-CL activity was determined spectrophotometrically in the presence of 50 μL of crude enzyme, 0.2 mM p-coumarate, 7.5 mM MgCl2, 0.8 mM ATP, and 38 mM CoA in 100 mM Tris-HCl buffer (pH 7.5), as described previously.25 One unit of 4-CL was defined as the amount of enzyme causing a decrease in A333 of 0.01 unit h−1. CHS activity was determined according to the method of Edwards and Kessman.26 F3H and DFR activities were measured using the method of Liu et al.,27 which determined the contents of F3H substrate (naringenin) and DFR substrate (dihydroquertin) with highperformance liquid chromatography (HPLC) analysis. UFGT activity was measured according to the method of Lister et al. with slight modification.28 The reaction mixture consisted of enzyme solution (100 μL), 50 mM bicine buffer (100 μL, pH 8.5), substrate (quercetin 15 μL of 2 mg/mL), and UDP-galactose (10 μL of 15 mg/mL). Reaction tubes were incubated at 30 °C for 30 min, and the reaction was terminated by adding 75 μL of 20% (v/v) trichloroacetic acid in methanol. The determination of ANS activity was based on that described previously.29 The reaction mixture (500 μL) consisting of 20 mM K-Pi (pH 7.0), 200 mM NaCl, 10 mM maltose, 5 mM DTT, 40 mM sodium ascorbate, 1 mM 2-oxoglutaric acid, 0.4 mM FeSO4, 1 M leucocyanidin, and ANS protein in 2 mg of crude cell extract was incubated at 30 °C for 30 min. The reaction was terminated by the addition of 5 μL of 36% (v/v) HCl, and the cyanidin formed was extracted by 500 μL of isoamyl alcohol for HPLC analysis. Cyanidin analysis was carried out by using a Waters 2695 HPLC equipped with a 2998 photodiode array detector (Waters Corp., Milford, MA, USA) according to the method of Saito et al.29 Protein content in the enzyme extracts was estimated using the Bradford method,30 using bovine serum albumin as a standard. Statistical Analysis. All statistical analyses were processed using SPSS (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance (ANOVA). The means were separated by Duncan’s multiple-range test, and differences at P < 0.05 were considered to be significant.
Previous literature showed that anthocyanin composition and anthocyanin contents in strawberries can be enhanced by ultraviolet radiation, altered gas composition, benzothiadiazole7-carbothioic acid S-methylester (BTH), or application of signaling molecules.14−18 Anthocyanin accumulation in response to different visible light wavelengths (e.g., blue, red, and green) has been investigated in strawberries during fruit development.14 However, no information is available on the effect of blue light on anthocyanin content in postharvest strawberry fruit. In this paper, blue LED light was applied to the postharvest strawberry fruit, and the effects on anthocyanin content and related enzymes of anthocyanin synthesis were explored.
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MATERIALS AND METHODS
Plant Materials. Strawberry (Fragaria ananassa Duch. cv. Fengguang) fruits were harvested by hand at stage of three-fourths colored based on the first appearance of red color on the fruit surface. Fruit were harvested and removed from a farm at Ningbo, Zhejiang province, and transported within 2 h to the laboratory. Fruits were selected for uniform size and color and then divided into two groups randomly. LED Blue Light Irradiation. The first group of strawberries was irradiated with blue (470 nm) light at an intensity of 40 μmol m−2 s−1 for 12 days at 5 °C (80−90% relative humidity); this dose was chosen in our present study on the basis of our preliminary research (data not shown). GreenPower LED Research Module Blue (Koninklijke Philips Electronics N.V., The Netherlands) was used in blue light irradiation, and the flux intensity of the LEDs at the level of samples was measured with a digital visible light meter (model 9.4 Blue Light, Solartech Inc., USA). The second group of fruit was stored at 5 °C in the dark (80− 90% relative humidity) and considered as the control. There were three replicates of 5 kg of fruit each per treatment, and the experiment was conducted twice. Samples were taken initially and at 2 day intervals during storage, immediately frozen in liquid nitrogen, and kept at −80 °C until required. Total Anthocyanin Content Determination. The pH differential method19 was applied to measure the total anthocyanin concentration of strawberry fruit. One gram samples from each replicate were homogenized with 5 mL of precooled acidified water (3% formic acid), and after centrifugation at 10000g for 15 min (4 °C), then another 5 mL of precooled acidified water was used to extract the residue again. The supernatant was combined to make the final volume of 25 mL for analysis. Results were expressed as milligrams of cyanidin-3-glucoside (Cy-3-Glu) equivalents per gram of fresh weight. Enzyme Assays. G6PDH and SKDH were extracted with 0.1 M potassium phosphate buffer (pH 7.4) containing 2 mM cysteine, 2 mM EDTA, and 0.5 mM dithiothreitol (DTT). PAL and TAL were extracted with 0.2 mM sodium borate buffer (pH 8.7), containing 20 mM β-mercaptoethanol. Fruit flesh (5 g) was ground with 200 mM Tris-HCl buffer (pH 7.5) containing 25% (v/v) glycerol and 0.1 M DTT for C4H, 4-CL, F3H, and DFR. The CHS extraction was carried out by grinding the tissue with 300 μL of 50 mM KH2PO4 (pH 8.0) containing 20 mM ascorbic acid. The mixture was ultrasonicated in ice for 2 min. For UFGT, the samples were suspended in the buffer solution, including 100 mM Tris-HCl (pH 7.5), 10 mM sodium ascorbate, 5 mM dithiothreitol, 0.1% β-mercaptoethanol (v/v), and 10 μM p-aminophenylmethanesulfonyl fluoride (p-APMSF). The extracts were homogenized at 4 °C and centrifuged (12000g, 15 min). The supernatants were used for enzyme assays. G6PDH activity was determined in reaction mixtures containing 5.88 μM NADP, 53.7 μM gluose-6-phosphate, and 88.5 mM MgCl2, which followed the method of Debham and Emes with slight modification.20 One unit of enzyme activity was equivalent to the oxidation of 1 μmol of NADPH per minute. SKDH activity was performed at 25 °C in the presence of 4 mM shikimic acid and 2.0 mM NADP in 0.1 mM Tris-HCl buffer at pH 9.0 as previously described.21
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RESULTS Effect of Blue Light Treatment on Total Anthocyanin Content in Strawberry Fruit. The total anthocyanin content was quantitatively determined in strawberry fruit over 12 days of storage (Figure 2). The concentration of total anthocyanin in the control group increased sharply during the first 4 days of storage, but did not consistently afterward. In contrast, the content in the blue light-treated strawberry fruit increased during the whole storage. As compared to the control fruit, the values of total anthocyanin concentration were significantly (p < 0.05) higher after 2 days of storage. Effect of Blue Light Treatment on G6PDH and SKDH Activities in Strawberry Fruit. G6PDH activity in both the control and blue light treatment increased during the first 8 days of storage and then declined (Figure 3A). SKDH activity increased in both blue light treatment and control samples as the storage progressed (Figure 3B). Blue light treatment enhanced the activities of both enzymes significantly (P < 0.05) after 2 days of storage. 4779
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4-CL activity increased in all strawberry fruits regardless of treatment at the first 2 days of storage and declined thereafter. The activity in the blue light-treated fruit was significantly (P < 0.05) higher than that in the control during the whole storage (Figure 4D). Effect of Blue Light Treatment on CHS and F3H Activities in Strawberry Fruit. CHS and F3H activities in control strawberry fruit began to increase after 4 days of storage. However, a significant enhancement of these two enzymes was found in the blue light-treated fruit, and the treatment maintained higher activities of CHS and F3H after 2 days of storage (Figure 5). Effect of Blue Light Treatment on DFR, ANS, and UFGT Activities in Strawberry Fruit. DFR activity in both control and blue light-treated fruits increased first and declined after 4 and 6 days of storage, respectively (Figure 6A). No significant changes of ANS and UFGT activities in control fruit were observed throughout the storage time. However, the activities of ANS and UFGT in blue light-treated fruit increased during the first 10 and 8 days of storage, respectively, and decreased afterward (Figure 6B,C). There was a positive effect of blue light treatment on all three enzymes whereby the treatment maintained significantly (P < 0.05) higher enzyme activities after 2 days of storage as compared to the control fruit.
Figure 2. Effect of blue light treatment on total anthocyanin content in strawberry fruit during storage at 5 °C. Values are means ± SE. Vertical bars represent standard errors of the means. Asterisks indicate significant differences between blue light-treated and control samples (Duncan’s multiple-range test; ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001).
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DISCUSSION Anthocyanins represent a group of natural flavonoid compounds in plants and are responsible for the coloration of strawberry fruit. Environmental conditions are known to induce anthocyanin accumulation across the major groups of higher plants, of these light being the best studied. It was reported that blue LED light regulated the metabolic pathways causing both increased concentrations of bioprotective compounds and plant growth of red leaf lettuce.31 Similar to the previous study at different development stages of strawberries,14 in our present work, we also found blue light treatment could enhance anthocyanin content in postharvest strawberries after 2 days of storage (Figure 2), which suggested that blue light could be a potential method to enhance anthocyanin accumulation and maintain a high-quality product of postharvest strawberry fruit. Anthocyanins are secondary metabolites, primarily synthesized via the pentose phosphate pathway and shikimate, phenylpropanoid, and flavonoid pathways. These pathways consist of a number of enzymatic steps, each catalyzed by a sequential reaction for anthocyanin synthesis (Figure 1). Therefore, in this study, we examined the possible role of key regulatory enzymes in anthocyanin metabolism in response to blue light treatment. G6PDH and SKDH are the crucial enzymes in the pentose phosphate and shikimate pathways, which are both involved in anthocyanin biosynthesis. CO2 stress enhanced phenolic accumulation in the root of Panax ginseng via induction of G6PDH activity. In addition, BTH treatment increased the activities of G6PDH and SKDH in postharvest strawberry fruit,14 which was responsible for the enhancement of anthocyanin biosynthesis. Similarly, in our present study, the increased anthocyanin content in blue lighttreated strawberry fruit was probably associated with the induction of these two enzymes as well. The phenylpropanoid pathway plays an important role in anthocyanin synthesis. The result of the coordinated action of many related enzymes in the phenylpropanoid pathway might be attributed to the accumulation of anthocyanin in
Figure 3. Effect of blue light treatment on G6PDH and SKDH activities in strawberry fruit during storage at 5 °C. Values are means ± SE. Vertical bars represent standard errors of the means. Asterisks indicate significant differences between blue light-treated and control samples (Duncan’s multiple-range test; ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001).
Effect of Blue Light Treatment on TAL, PAL, C4H, and 4-CL Activities in Strawberry Fruit. TAL and PAL activities in both control and blue light treatment increased during the initial 6 days of storage; thereafter they declined gradually (Figure 4A,B). In comparison with the control berries, significantly (P < 0.05) higher activities of TAL and PAL were observed in blue light-treated fruit after 4 and 2 days of storage, respectively. C4H activity in the control strawberry fruit increased gradually in the first 6 days and then decreased over the next 6 days. On the other hand, the activity in blue light-treated samples increased rapidly, reached a maximum level at 8 days, and then declined afterward (Figure 4C). When compared to the control fruit, blue light treatment maintained significantly (P < 0.05) higher C4H activity after 6 days of storage. 4780
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Figure 4. Effect of blue light treatment on TAL, PAL, C4H, and 4-CL activities in strawberry fruit during storage at 5 °C. Values are means ± SE. Vertical bars represent standard errors of the means. Asterisks indicate significant differences between blue light-treated and control samples (Duncan’s multiple-range test; ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001).
present study, the higher PAL and TAL activities as well as the increased activities of C4H and 4-CL observed in blue lighttreated strawberries indicated that an enhancement in anthocyanin production by blue light was accompanied by induction of the four key enzymes in the phenylpropanoid pathway of strawberry fruit. Anthocyanin accumulation is also thought to be the result of interaction of the multiple key enzymes in the flavonoid pathway.33 An increase of enzyme activities or gene expression involved in this pathway along with an increase in anthocyanin accumulation in response to light exposure has been reported. For example, Ju et al. have shown in ‘Fuji’ apples that lightreflecting mulches increased anthocyanin concentration and UFGT activity.34 Ubi et al. demonstrated that increase in the expression levels of all five biosynthetic genes, CHS, F3H, DFR, ANS, and UFGT, in fruit skin of five apple cultivars coincided with increases of anthocyanin concentration.35 When the fruit was bagged about 1 month prior to commercial harvest to prevent anthocyanin biosynthesis, the expression of CHS, ANS, and UFGT was substantially depressed in fruit skin, whereas that of all five genes was enhanced by UV-B treatment with the accumulation of anthocyanins.35 Our results agreed with these authors because we found that blue light enhanced the activities of CHS, F3H, DFR, ANS, and UFGT in postharvest strawberries during storage, resulting in anthocyanin biosynthesis.11 Previous study has revealed that blue light induced anthocyanin accumulation in strawberries during fruit development by regulating the expression of anthocyanin biosynthetic genes. Together with this study, our results suggested that blue light regulated multiple key enzymes in the anthocyanin pathway, thereby enhancing anthocyanin biosynthesis in strawberries during both fruit development and postharvest storage; however, the possible differences in regulatory mechanisms of anthocyanin accumulation by blue light applied before or after harvest need further study. In conclusion, blue light was effective for stimulating anthocyanin accumulation in postharvest strawberry fruit. The
Figure 5. Effect of blue light treatment on CHS and F3H activities in strawberry fruit during storage at 5 °C. Values are means ± SE. Vertical bars represent standard errors of the means. Asterisks indicate significant differences between blue light-treated and control samples (Duncan’s multiple-range test; ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001).
strawberries after blue light treatment. Therefore, the four pivotal enzymes involved in this pathway, PAL, TAL, C4H, and 4-CL, were investigated. It has been shown that treatment with gibberellic acid increased PAL and TAL activities, which was correlated with the anthocyanin accumulation in ripening strawberries.32 BTH induced anthocyanin content in postharvest strawberry fruit by enhancing activities of the four enzymes of phenylpropanoid metabolism.14 Thus, in our 4781
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REFERENCES
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Figure 6. Effect of blue light treatment on DFR, ANS, and UFGT activities in strawberry fruit during storage at 5 °C. Values are means ± SE. Vertical bars represent standard errors of the means. Asterisks indicate significant differences between blue light-treated and control samples (Duncan’s multiple-range test; ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001).
enhancement in anthocyanin content by blue light might result from the activation of key enzymes in the pentose phosphate, shikimate, phenylpropanoid, and flavonoid pathways. Blue light could be considered as a supplemental light source in postharvest strawberry fruit to improve levels of healthpromoting compounds, especially anthocyanin accumulation.
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AUTHOR INFORMATION
Corresponding Author
*(Z.Y.) Phone:+86-574-88222229. Fax: +86-574-88222991. Email:
[email protected]. Funding
This study was supported by the National Science Foundation of China (31101356), the Natural Science Foundation of Ningbo City (2013A610155, 2013A610159), the Induction of Talent Project (ZX2012000031), and the K. C. Wong Magna Fund at Ningbo University. Notes
The authors declare no competing financial interest. 4782
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