Mechanism Underlying the Onset of Internal Blue Discoloration in

Aug 16, 2016 - Katsunori Teranishi†, Nagata Masayasu‡, and Daisuke Masuda§. † Graduate School of Bioresources, Mie University, 1577 Kurimamachi...
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Mechanism Underlying the Onset of Internal Blue Discoloration in Japanese Radish (Raphanus sativus) Roots Katsunori Teranishi,*,† Nagata Masayasu,‡ and Daisuke Masuda§ †

Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan § Sand Dune Agricultural Research Center, Agricultural Experiment Station, Ishikawa Agriculture and Forestry Research Center, I 5-2 Uchihisumi, Kahoku, Ishikawa 929-1126, Japan ‡

S Supporting Information *

ABSTRACT: The internal blue discoloration observed in Japanese radish (Raphanus sativus L.) roots is a physiological phenomenon caused by storage following harvest at approximately 20 °C and poses a serious problem for farmers. Here, we describe the mechanism underlying the onset of internal blue discoloration of three cultivars: Hukuhomare, SC8-260, and Yuto. Each cultivar was maintained under the same conditions. Additionally, Hukuhomare radish roots were maintained at three different cultivation conditions in a related experiment. The blue discoloration in radish roots was caused by the oxidation of 4-hydroxyglucobrassicin as a result of an increase in oxidative stress involving peroxidase. Thus, the extent of blue discoloration was influenced by the chemical balance involving 4-hydroxyglucobrassicin content, antioxidant capacity, and oxidation activity. KEYWORDS: Raphanus sativus, Japanese radish, roots, blue discoloration, 4-hydroxyglucobrassicin

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INTRODUCTION Internal blue discoloration in Japanese radish (Raphanus sativus L.) is a physiological phenomenon that usually occurs in harvested radish roots after storage at approximately 20 °C for a few days. Blue components are produced in the roots, and recently, its onset in some radish cultivars has been reported to be increasing in Japan. Because the discoloration decreases the commercial value of the affected radishes as a result of the strange appearance of blue color, it represents a serious issue for farmers. The incidence of this type of discoloration is dependent upon the type of radish cultivar (Figure 1B) and the cultivation conditions (Figure 2A).1 In addition, the incidence of blue discoloration is specific to particular regions of the plant, and it affects the xylem at approximately 20−150 mm from the root tip. Recently, we developed a rapid and simple method based on soaking freshly harvested radish roots in an aqueous solution of H2O2 at room temperature to artificially generate the blue discoloration (Figure 1C and Figure 2B).2 The resultant discoloration can then be visually assessed. Many previous studies have focused on the physiological mechanism causing brown discoloration in radish roots.3−6 However, there have been few studies regarding blue discoloration; additionally, its causes and underlying mechanisms remain unclear. To circumvent the blue discoloration after harvest, it is essential to develop new radish cultivars that do not show discoloration and/or to improve the methods of cultivation. Thus, understanding the mechanisms causing blue discoloration could generate solutions to this issue. Here, the mechanisms underlying the onset of blue discoloration are investigated. In our previous study, the blue components did not show the characteristics of anthocyanins, including pH-dependent color changes and resistance to reduction by ascorbic acid,2 and we demonstrated through high-performance liquid © 2016 American Chemical Society

chromatography−photodiode array (HPLC−PDA) analysis that chromatographic characteristics showed existence of many blue components and did not differ between blue components extracted from radish roots stored at 20 °C for 4 days and those generated by treating fresh radish root sections with aqueous H2O2 solution.2 These results suggest that blue discoloration during storage at 20 °C could be linked to the oxidation of components in radish roots. Additionally, using an assay system with H2O2 and peroxidase, a precursor to the blue components was successfully isolated from Hukuhomare radish roots, which are known to be affected by blue discoloration in Japan. The precursor to the blue components was identified as 4-hydroxyglucobrassicin,2 a compound commonly found in seeds, sprouts, and roots of Brassica plants.7,8 On the basis of the results by Truscott et al.,7,9−11 we suggested that the blue components possess an indol-(4,7)-p-quinone skeleton, which is likely produced via the blue discoloration reaction.2 In the present study, the mechanisms underlying the onset of blue discoloration during storage at 20 °C were elucidated using three cultivars of Japanese radish: Hukuhomare, SC8-260, and Yuto. Each was cultivated under similar conditions; additionally, Hukuhomare radish roots were cultivated in three different settings.

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MATERIALS AND METHODS

Plant Materials and Cultivation Conditions. Hukuhomare, SC8-260, and Yuto radish seeds were purchased from Mikado Kyowa Seed Co., Ltd., Sakata Seed Co., Ltd., and Nanto Seed Co., Ltd., in Japan, respectively. These radish cultivars were grown in the Ishikawa

Received: Revised: Accepted: Published: 6745

May 11, 2016 August 14, 2016 August 16, 2016 August 16, 2016 DOI: 10.1021/acs.jafc.6b02103 J. Agric. Food Chem. 2016, 64, 6745−6751

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Journal of Agricultural and Food Chemistry Sand Dune Agricultural Research Center field site in 2012 under similar cultivation conditions. In 2013, Hukuhomare radish plants were grown in three independent plots (Kanazawa, Hakui, and Kahoku) exposed to different cultivation conditions. The plants were sown on September 5th, and roots were harvested 60 days after sowing. Extraction. Freshly harvested Hukuhomare, SC8-260, and Yuto radish roots were cut vertically along the middle axis, and 10 × 10 × 10 mm xylem sections were extracted at 50, 100, and 150 mm from the root tip. Xylem sections of Hukuhomare radish cultivars cultivated at Kanazawa, Hakui, and Kahoku were also extracted by following a similar procedure. Each section was homogenized using MeOH (1 mL) in an ice bath, and the homogenate was filtered through absorbent cotton. The resultant filtrate was centrifuged at 10000g at 0 °C for 5 min, and the supernatant was filtered through 0.45 μm membrane filters. The resultant filtrate, designated “extract solution”, was stored at −80 °C until further use. Fresh Hukuhomare roots cultivated at Hakui were stored at 20 °C and approximately 80% relative humidity for 0, 4, and 6 days in the dark before 10 × 10 × 10 mm xylem sections were extracted at 50 mm from the root tip. Extract solutions were prepared as described above. Quantification of 4-Hydroxyglucobrassicin and Ascorbic Acid. To quantify the 4-hydroxyglucobrassicin and ascorbic acid contents in the extract solutions, an HPLC−PDA−mass spectrometry (MS) spectrometer equipped with a JASCO Gulliver HPLC system, MD-910 detector (JASCO Corp., Tokyo, Japan), and Cosmosil 5C18-PAQ column (4.6 × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) was used. The mobile phase was a mixture of aqueous 0.1% (v/v) trifluoroacetic acid (TFA) solution (A) and 0.1% (v/v) TFA/MeOH solution (B). The flow rate was 0.8 mL/min in a linear gradient starting with 0% B and reaching 50% B in 20 min. 4-Hydroxyglucobrassicin was identified using a ZQ 4000 MS spectrometer (Waters Corporation, Milford, MA) in the electrospray ionization (ESI) negative mode. 4-Hydroxyglucobrassicin and ascorbic acid were detected by monitoring the absorbance at 250 nm. Concentrations of 4-hydroxyglucobrassicin and ascorbic acid in the extract solutions were quantified using calibration curves of chemically synthesized 4-hydroxyglucobrassicin2 and commercially available ascorbic acid; their content in the sections is provided as the mean ± standard deviation (SD) of six independent measurements and expressed as nanomoles of 4-hydroxyglucobrassicin and ascorbic acid equivalents per gram of fresh roots. Total Antioxidant Capacity Assay. The total antioxidant capacity of the extract solutions (prepared as described in the above Extraction section) was determined as the amount of Cu2+ reduced to Cu+ using a total antioxidant capacity colorimetric assay kit (BioVision, Inc., Milpitas, CA) according to the instructions of the manufacturer. Briefly, extract solutions (3.3 μL) and distilled water (96 μL) were placed into individual wells of a 96-well plate before adding Cu2+ reagent solution (100 μL). The plate was incubated at 25 °C for 1.5 h, and the absorbance at 570 nm was measured using a microplate absorbance reader (Sunrise Rainbow, Tecan Japan Co., Ltd., Kanagawa, Japan). Trolox standard was used to build the calibration curve. The results are provided as the mean ± SD of six independent measurements, and total antioxidant capacity is expressed as Cu2+ content reduced to Cu+. Visualization of Peroxidase Activity in Radish Roots. The guaiacol method was employed to visualize peroxide activity in radish roots.12,13 Freshly harvested Hukuhomare radish roots as well as Hukuhomare radish roots stored at 20 °C at approximately 80% relative humidity for 4 days in the dark were vertically cut along the middle axis. An aqueous solution containing H2O2 (0.14 M) and o-methoxyphenol (4 mM) was added to homogeneously cover the section surface at room temperature. After 5 min of incubation, the resultant browning was visually assessed. Treatment of Radish Root Sections with Sodium Azide. Freshly harvested Hukuhomare radish roots cultivated at Kanazawa were cut in round slices at 50 mm from the root tip. The sections were soaked in distilled water or 0.1 or 1 M aqueous sodium azide solution at room temperature for 10 min. After removal of the sodium azide solution, the sections were lightly rinsed with distilled water. Then, the

Figure 1. Blue discoloration of freshly harvested radish roots from Hukuhomare, SC8-260, and Yuto cultivars: (A) normal root sections, (B) after storage at 20 °C for 4 days, and (C) after soaking in 0.29 M aqueous H2O2 solution at room temperature for 10 min. sections were soaked in 0.29 M aqueous H2O2 solution at room temperature for 10 min and observed for discoloration. To generate a slight blue discoloration, freshly harvested Hukuhomare radish roots cultivated at Kanazawa were stored at 20 °C and approximately 80% relative humidity in the dark for 3 days. Then, the radish roots were cut vertically along the middle axis, and the sections were soaked in distilled water or 0.1 or 1 M aqueous sodium azide solution and incubated at room temperature for 10 min. After removal of the sodium azide solution, the sections were slightly rinsed using distilled water. These sections were kept at 20 °C for 5 h in a dark box to maintain the humidity prior to visually assessing the level of discoloration. Chemical Structure Determination of the Precursor to Blue Components in SC8-260 Radish Roots. Extraction, isolation, and blue discoloration assays were conducted according to the method by Teranishi and Nagata.2 The isolated precursor to blue components in SC8-260 radish roots was identified by HPLC−PDA−MS using the JASCO Gulliver HPLC system with a MD-910 detector (220− 650 nm), a ZQ 4000 MS spectrometer, and a Cosmosil 5C18-PAQ column (4.6 × 250 mm). The mobile phase was a mixture of aqueous 6746

DOI: 10.1021/acs.jafc.6b02103 J. Agric. Food Chem. 2016, 64, 6745−6751

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Figure 2. Blue discoloration of freshly harvested Hukuhomare radish roots from the Kanazawa, Hakui, and Kahoku producing districts observed (A) after storage at 20 °C for 4 days and (B) after soaking in 0.29 M aqueous H2O2 solution for 10 min.

Figure 4. Contents of (A) 4-hydroxyglucobrassicin, (B) ascorbic acid, and (C) total antioxidant capacity in xylem sections obtained at 50, 100, and 150 mm from the root tip of freshly harvested Hukuhomare, SC8-260, and Yuto radish roots (n = 6). (∗) Below the detection limit. 0% B and reaching 50% B in 20 min. MS analyses were conducted for both ESI positive and negative modes. Reaction of Blue Components with Ascorbic Acid. A total of 1 mmol/L 4-hydroxyglucobrassicin (0.5 mL) was added to a micro quartz cell before measuring the absorption spectrum at 20 °C using a V-530 DS spectrometer. A total of 0.29 mol/L aqueous hydrogen peroxide (7 μL) and 1500 units/mL horseradish peroxidase (HRP) solution (30 μL) were added to the solution at 20 °C to generate blue components. After 4 h, the ultraviolet−visible (UV−vis) absorption spectrum was measured. Then, 1 mol/L aqueous ascorbic acid (3 μL) was added at 20 °C, and the UV−vis absorption spectrum was measured after 10 min.

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RESULTS AND DISCUSSION In our preliminary study, 18 cultivars of Japanese radish roots were stored at 20 °C for 4 days or soaked in 0.29 M aqueous H2O2 solution at room temperature for 10 min to generate blue discoloration (Figure S1 of the Supporting Information). The collected data were used to understand the relation between the degree of discoloration obtained by the two treatments and the type of radish cultivar. It was found that discoloration in Hukuhomare radish roots was more evident than that in other radish types for both roots stored at 20 °C and those treated with H2O2 (Figure 1). In addition, almost none of the SC8-260 radish roots showed any symptoms of discoloration after storage at 20 °C; however, they showed

Figure 3. HPLC chromatograms of extracts obtained from (A) freshly harvested SC8-260 and (B) Yuto radish roots. The arrow indicates the peak in chromatogram A corresponding to the precursor of the blue components. 0.1% (v/v) TFA solution (A) and 0.1% (v/v) TFA/MeOH solution (B). The flow rate was 0.8 mL/min in a linear gradient starting from 6747

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Figure 6. Contents of (A) 4-hydroxyglucobrassicin, (B) ascorbic acid, and (C) total antioxidant capacity in xylem sections obtained at 50 mm from the root tip of Hukuhomare radishes stored at 20 °C and (D) Hukuhomare radish stored at 20 °C for 6 days. (∗) Below detection limits.

Figure 5. Contents of (A) 4-hydroxyglucobrassicin, (B) ascorbic acid, and (C) total antioxidant capacity in xylem sections obtained at 50 mm from the root tip of freshly harvested Hukuhomare radish roots cultivated at Kanazawa, Hakui, and Kahoku (n = 6).

moderate levels of discoloration following treatment with H2O2 (Figure 1). For those roots prone to blue discoloration, the lower sections of the roots exhibited stronger discoloration than the upper parts. Conversely, Yuto radish roots showed little or no discoloration after both storage at 20 °C and treatment with H2O2 (Figure 1). For Hukuhomare radish roots cultivated under three different field conditions (Kanazawa, Hakui, and Kahoku), significant differences were found in the degree of discoloration among the field cultivation conditions, as shown in Figure 2. Set against this background, the mechanisms underlying the onset of this distinctive blue discoloration were investigated. In our previous study, it was found that 4-hydroxyglucobrassicin was the only precursor to the blue components in Hukuhomare roots.2 In the present study, the precursor to the blue components was isolated from SC8-260 radish roots using HPLC following discoloration with H2O2 and HRP, as described for Hukuhomare radish roots (Figure 3). In this case, 4-hydroxyglucobrassicin was identified again as the sole precursor to the blue components; however, the precursor to the blue components in Yuto radish roots was not identified (Figure 3).

Figure 7. Loss of the blue coloration generated by 4-hydroxyglucobrassicin treated with hydrogen peroxide and HRP, as a consequence of the action of ascorbic acid.

Figure 4 shows 4-hydroxyglucobrassicin content in xylem sections at 50, 100, and 150 mm from the root tip from freshly harvested Hukuhomare, SC8-260, and Yuto radish roots. In Hukuhomare and SC8-260 roots, the 4-hydroxyglucobrassicin content in the lower sections was greater than that in the upper sections. In addition, the 4-hydroxyglucobrassicin content in Hukuhomare 6748

DOI: 10.1021/acs.jafc.6b02103 J. Agric. Food Chem. 2016, 64, 6745−6751

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Figure 8. Effect of sodium azide on blue discoloration of Hukuhomare radish roots (A) treated with H2O2 and (B) stored at 20 °C.

present any 4-hydroxyglucobrassicin, which followed the lack of discoloration after storage at 20 °C or after treatment with H2O2. Moreover, as shown in Figure 5A, the 4-hydroxyglucobrassicin content in xylem sections at 50 mm from the root tip of freshly harvested Hukuhomare radish roots cultivated under three different

radish roots was directly related to the degree of blue discoloration observed after both storage at 20 °C and treatment with H2O2 (panels B and C of Figure 1); however, this relation was only found in roots treated with H2O2 for SC8-260 radishes (panels B and C of Figure 1). Yuto radish roots did not 6749

DOI: 10.1021/acs.jafc.6b02103 J. Agric. Food Chem. 2016, 64, 6745−6751

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Journal of Agricultural and Food Chemistry field conditions corresponded with the degrees of blue discoloration observed in roots stored at 20 °C and treated with H2O2, as shown in Figure 2. Therefore, these results showed a direct link between the degree of blue discoloration and 4-hydroxyglucobrassicin content in these radish roots, with the exception of SC8-260 radish roots stored at 20 °C. In our previous study, we demonstrated a similarity between HPLC−PDA characteristics of the blue components from Hukuhomare radish roots stored at 20 °C and those of blue components from Hukuhomare radish roots treated with H2O2. This suggested that oxidation of 4-hydroxyglucobrassicin might be responsible for the blue discoloration observed after storage at 20 °C. Consequently, we believed that reductive components, such as ascorbic acid, present in radish roots could also influence the onset of blue discoloration. Therefore, the relation between blue discoloration, content of ascorbic acid, and total antioxidant capacity in fresh root sections was investigated. The content of ascorbic acid (Figure 4B) and the total antioxidant capacity (Figure 4C) of SC8-260 radish xylem sections at 50, 100, and 150 mm from the root tip were significantly higher than those of Hukuhomare roots (panels B and C of Figure 4). The presence of H2O2 in excess leads to the oxidation of reductive components, such as ascorbic acid, amino acids, and proteins; consequently, reductive components might be able to inhibit the onset of blue discoloration linked to 4-hydroxyglucobrassicin oxidation. Therefore, it was tested whether Hukuhomare and SC8-260 radish roots treated with H2O2 showed a greater degree of blue discoloration than storage at 20 °C and whether this discoloration was linked to the 4-hydroxyglucobrassicin content and reductive capacity within the roots. SC8-260 radish roots stored at 20 °C contained enough 4-hydroxyglucobrassicin to generate the blue discoloration (although the content was lower than that of Hukuhomare radish roots); their higher antioxidant capacity compared to that in Hukuhomare radish roots efficiently suppressed blue discoloration. The antioxidant capacity did not differ significantly among field cultivation conditions for Hukuhomare radish roots after storage at 20 °C (panels B and C of Figure 5), suggesting that the blue discoloration depended only upon the 4-hydroxyglucobrassicin content. The 4-hydroxyglucoburassicin content significantly decreased over time in Hukuhomare roots compared to that in fresh roots, as shown in Figure 6A. These results suggest that 4-hydroxyglucobrassicin decreased with the degree of discoloration over time. The ascorbic acid content also declined over time (Figure 6B), while total antioxidant capacity did not show a significant time-dependent decrease (Figure 6C). These results suggested that ascorbic acid was consumed as a consequence of the oxidative stress induced during storage at 20 °C; however, total antioxidant capacity remained constant to facilitate resistance to oxidative stress, which compensated for the loss in ascorbic acid. Moreover, it was shown that ascorbic acid decolorized blue components generated from 4-hydroxyglucobrassicin with H2O2 and peroxidase (Figure 7). Thus, it is not unlikely that other reductive components could also decolorize the blue components. To this end, the decrease in ascorbic acid observed following storage at 20 °C could be linked to the degree of decoloration and the resistance to oxidative stress. In fact, the blue component content appeared to decrease after 6 days (Figure 6D). In our previous study, it was shown that a combination of H2O2 and peroxidase leads to blue discoloration with 4-hydroxyglucobrassicin,2 supporting the role of intracellular peroxidase in the discoloration. This mechanism was confirmed

by inhibiting peroxide activity with azide compounds. As shown in Figure 8A, treating freshly harvested Hukuhomare radish root sections with sodium azide prior to the addition of H2O2 significantly suppressed the progression of blue discoloration induced by H2O2. However, it should be noted that blue components generated using H2O2 did not fade after the addition of sodium azide. For the experiments using radish roots at the early stages of blue discoloration following storage at 20 °C, sodium azide inhibited further blue discoloration (Figure 8B), suggesting the involvement of peroxidase in blue discoloration of roots stored at 20 °C. Our subsequent efforts to measure peroxidase activity in extract solutions using colorimetric and chemiluminescence methods failed because the addition of H2O2 as a reagent for the peroxidase assay caused interference in the assay. The peroxidase activity on whole root surfaces was quantitatively visualized using the guaiacol method. Visual assessment of the peroxidase activity in fresh Hukuhomare radish roots and roots stored at 20 °C for 4 days showed a uniform distribution throughout the roots; in fact, peroxide activity was not necessarily found in larger concentrations in the regions showing larger degrees of discoloration (Figure 9).

Figure 9. Visualization of peroxidase activity in (A) fresh Hukuhomare radish roots and (B) roots stored at 20 °C for 4 days visualized using the guaiacol method. Slightly black discoloration at the xylem at approximately 20−150 mm from the root tip is due to blue discoloration by reaction with H2O2.

Thus far, any significant amount of H2O2 or other active oxygen species has not been detected in roots at the discoloration stage. In radish roots, catalase, superoxide dismutase, and reductive components, such as ascorbic acid, immediately suppress active oxygen species, as does 4-hydroxyglucobrassicin as a naturally reductive component. As a consequence, it is difficult to detect the actual degree of oxidative stress experienced at the discoloration stage (before suppression by antioxidants). In conclusion, the onset of blue discoloration following storage at 20 °C depended upon the 4-hydroxyglucobrassicin content, oxidative stress involving peroxidase, and antioxidant capacity of radish roots. Thus, radish roots with low 4-hydroxyglucobrassicin contents and/or a larger reductive component and/or low peroxidase activity would be desirable to suppress the onset of blue discoloration. At present, we are aiming to develop novel cultivars with no blue discoloration and cultivation methods capable of suppressing blue discoloration based on these results. It is desired to successfully develop a discoloration-free radish and/or cultivation method in the near future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02103. Three groups of blue discoloration from 18 cultivars of Japanese radish roots (Figure S1) (PDF) 6750

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +81-59-231-9615. E-mail: [email protected]. Funding

Katsunori Teranishi gratefully acknowledges financial support from the Adaptable and Seamless Technology Transfer Program through target-driven research and development, Japan Science and Technology Agency (JST) (AS251Z00176N), and the Towa Foundation for Food Science & Research. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. H. Imaseki, Professor Emeritus, Nagoya University, for his valuable suggestion for the start of this research.

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REFERENCES

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DOI: 10.1021/acs.jafc.6b02103 J. Agric. Food Chem. 2016, 64, 6745−6751