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Distribution analysis of anthocyanins, sugars, and organic acids in strawberry fruits using matrix-assisted laser desorption/ionization-imaging mass spectrometry Hirofumi Enomoto, Kei Sato, Koji Miyamoto, Akira Ohtsuka, and Hisakazu Yamane J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00853 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Journal of Agricultural and Food Chemistry

Distribution analysis of anthocyanins, sugars, and organic acids in strawberry fruits using matrix-assisted laser desorption/ionization-imaging mass spectrometry

Hirofumi Enomoto,*,†,‡,§ Kei Sato,‡ Koji Miyamoto,†,‡ Akira Ohtsuka,┴ and Hisakazu Yamane†,§

†

Department of Biosciences, Faculty of Science and Engineering, Teikyo University, Utsunomiya 320-8551, Japan

‡

Division of Integrated Science and Engineering, Graduate School of Science and Engineering, Teikyo University, Utsunomiya 320-8551, Japan

§

Advanced Instrumental Analysis Center of Teikyo University, Teikyo University, Utsunomiya 320-8551, Japan

┴

Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

Corresponding Author *(H. E.) Phone: +81 28 627 7312 Fax: +81 28 627 7187 E-mail: [email protected]

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ABSTRACT: Anthocyanins, sugars, and organic acids contribute to the appearance,

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health benefits, and taste of strawberries. However, their spatial distribution in the ripe

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fruit has been fully unrevealed. Therefore, we performed matrix-assisted laser

4

desorption/ionization (MALDI)-IMS analysis to investigate their spatial distribution in

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ripe strawberries. The detection sensitivity was improved by using the TM-Sprayer for

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matrix application. In the receptacle, pelargonidins were distributed in the skin, cortical,

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and pith tissues, whereas cyanidins and delphinidins were slightly localized in the skin. In

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the achene, mainly cyanidins were localized in the outside of the skin. Citric acid was

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mainly distributed in the upper and bottom side of cortical tissue. Although hexose was

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distributed almost equally throughout the fruits, sucrose was mainly distributed in the

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upper side of cortical and pith tissues. These results suggest that using the TM-Sprayer in

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MALDI-IMS was useful for microscopic distribution analysis of anthocyanins, sugars,

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and organic acids in strawberries.

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KEYWORDS: Strawberry fruits; anthocyanins; sugars; organic acids; imaging mass

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spectrometry (IMS); matrix assisted laser desorption/ionization (MALDI)

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INTRODUCTION

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Either as a fresh fruit or in any form of processed presentation, strawberries (Fragaria

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× ananassa) are the most widespread and consumed berries around the world because of

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their attractive appearance, delicious taste, and health-related properties.1-6 Strawberries

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are rich in nutritious compounds including sugars, vitamins and organic acids, as well as

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in non-nutritious phytochemicals, especially polyphenols.7–10 Anthocyanins are the major

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polyphenolic compounds present in strawberries. Pelargonidin and cyanidin derivatives

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are the most representative and important ones, because they are responsible for the

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typical red color of the fruit.7,8,10 Several studies have shown that in addition to other

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classes of metabolites in strawberries, anthocyanins have

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anti-inflammatory activities; they are thus considered to be associated with the prevention

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of chronic pathologies, such as certain types of cancers 2−7,10 The contents and balance of

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sweetness and sourness are important factors that define the taste of strawberries. The

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predominant sweet metabolites are sugars, especially glucose, fructose, and sucrose,

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whereas the predominant sour metabolites are organic acids, especially citric and malic

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acids.7–9 However, the spatial localization of these metabolites in the body of the

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strawberry fruit has not been fully revealed.

antioxidative and

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In the routine analysis of metabolites in foods, modern separation techniques, such as

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liquid chromatography (LC) and in combination with mass spectrometry (MS), are

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used.11–13 These techniques are useful for qualitative and quantitative analysis of

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metabolites, and are also used to investigate their spatial distributions by dividing

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biological samples into different tissues; however, the resolution is dependent on the

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accuracy of sampling and tissue differentiation.14,15 To accomplish this task, imaging mass

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spectrometry (IMS) is an emerging technique that can be used to simultaneously 3 ACS Paragon Plus Environment


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investigate both content and spatial distribution of biomolecules from metabolites to

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proteins in biological tissues, without requiring antibodies, staining, or complicated

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preliminary procedures.16–18 Recently, IMS has been adapted in food analysis for

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identification and quantitation of metabolites, proteins, and pesticides in food products,

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which have been successfully visualized by using soft ionization techniques, such as

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matrix-assisted laser desorption/ionization (MALDI)19–26 or desorption electrospray

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ionization (DESI).27–30

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In MALDI-IMS, an organic, crystalline compound, namely the matrix, is applied on

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the biological tissue to assist analyte desorption and ionization. Matrix application is

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important technique because characteristics of the matrix such as the crystal size and

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amount affect the quality of the obtained mass spectral imaging, including the spatial

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resolution and detection sensitivity.31,32 Thus far, several methods have been developed

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for matrix application in MALDI-IMS to improve the matrix quality. The airbrush method

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has been commonly used for matrix application in MALDI-IMS. This method is

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relatively simple, fast, and cost-effective;20–22,25,26 however, it is difficult to control the

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spray rate strictly. Thus, the quality of the spray is extremely dependent on the user, and

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results are frequently not reproducible from one laboratory to another. Automatic matrix

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sprayer devices, such as the TM-Sprayer, have been developed to solve this problem with

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manual airbrush application. These devices can control some factors such as the speed of

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the matrix-spraying nozzle, which affect the mass imaging quality.31 Therefore, using

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automatic sprayer devices, the applied matrix crystal size and density on biological tissues

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is more uniform, and mass imaging results with higher reproducibility can be obtained.

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In the present study, we compared the detection sensitivity of MALDI-IMS analysis for

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food-related metabolites such as anthocyanins, sugars, and organic acids in ripe 4 ACS Paragon Plus Environment

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‘Tochiotome’ strawberries, a popular strawberry in Japan, by using the airbrush and

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TM-Sprayer techniques. Subsequently, we analyzed the spatial distribution of these

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metabolites in ripe strawberries.

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MATERIALS AND METHODS

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Reagents. Methanol and water were purchased from Sigma Aldrich (St. Louis, MO,

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USA). Carboxymethyl cellulose (CMC) sodium salt and formic acid (FA) were purchased

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from Wako Chemicals (Tokyo, Japan). 2,5-Dihydroxybenzoic acid (DHB) was purchased

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from Tokyo Kasei Co. (Tokyo, Japan). All reagents and solvents used in the present study

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were of analytical grade.

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Strawberry (Fragaria × ananassa Duch.) samples. The ‘Tochiotome’ strawberry

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fruits were cultivated in the Strawberry Research Center (Tochigi, Japan). About 30

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strawberries were harvested in winter after ripening and stored at −80 °C until use.

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Strawberry Metabolite Extraction. Fresh strawberries were freeze-dried and then

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finely homogenized. Homogenates were suspended in 50% methanol and kept under

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continuous shaking at room temperature for 2 h. The solutions were centrifuged at 3000

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rpm for 15 min, and the supernatants were collected as metabolite extracts. Extracts were

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analyzed by MALDI-MS and MALDI-tandem mass spectrometry (MS/MS) to identify

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the metabolites.

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MALDI-MS and MS/MS Analysis of Metabolites in Strawberry Extracts.

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Strawberry extracts (1 µL) were mixed with an equal volume of 40 mg/mL DHB in 70% 5 ACS Paragon Plus Environment


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aqueous methanol containing 0.1% FA. The mixed solution was deposited onto an indium

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tin oxide (ITO)-coated glass slide (Bruker Daltonics, Billerica, MA, USA) and dried

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before MALDI-MS analysis. MALDI-MS analysis was performed using a MALDI time

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of flight (TOF)/TOF-type instrument (UltrafleXtreme, Bruker Daltonics) equipped with a

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355-nm Nd:YAG laser at a repetition rate of 1000 Hz in positive-ion mode (reflector

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mode). Ions with m/z values in the range of 100–600 were measured. The m/z values were

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calibrated externally using the exact m/z of CHCA [M+H]+ ions (m/z 190.04987) and

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bradykinin (1-7) (Bruker Daltonics) [M+H]+ ions (m/z 757.39916).

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For MS/MS analysis, the selected precursor ions and the product ions were obtained by

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UltrafleXtreme in collision-induced dissociation “LIFT” MS/MS mode. The obtained

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MS/MS spectra were analyzed using Flexanalysis 3.4 software (Bruker Daltonics). The

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sugar moieties of anthocyanin species were identified from their neutral losses and

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previous reports10.

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Preparation of the Fruit Sections for MALDI-IMS. Fruit sections were prepared

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according to our previous study.27 Briefly, the fresh fruits were immersed in 2% CMC and

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flash-frozen in liquid nitrogen. Subsequently, longitudinal and cross-sections,

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80-µm-thick, were consecutively prepared using a CM 1860 cryostat (Leica

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Microsystems, Wetzlar, Germany). The sections were then mounted onto ITO-coated

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glass slides, which were dried in a vacuum desiccator for 20 min. The sections were

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placed in 50-ml conical centrifuge tubes that contained silica gel for drying and were

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preserved at -80 °C until MALDI-IMS analysis.

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Matrix Application. Matrix application was performed using two different techniques: 6 ACS Paragon Plus Environment

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airbrush with a 0.18-mm nozzle (Mr. Airbrush custom; Mr. Hobby, Tokyo, Japan) and the

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TM-Sprayer system (HTX Technologies, LLC, Carrboro, NC, USA). In the airbrush

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method, 1 mL of 40 mg/mL DHB solution in 70% aqueous methanol containing 0.1% FA

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was used. The distance between the sample and spray nozzle of the airbrush was kept at

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10 cm. In the TM-sprayer method, 30 mg/mL DHB solution in 50% aqueous methanol

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containing 0.1% FA was used. The solvent flow rate, nozzle speed, number of passes, and

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temperature were set at 0.2 mL/min, 1200 mm/min, 8, and 85 °C, respectively.

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MALDI-IMS Analysis. IMS was performed according to a previous study33, with

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some modification. A MALDI TOF/TOF-type mass spectrometer (UltrafleXtreme, Bruker

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Daltonics) was used. Data were acquired with a step size of 200 µm. The laser diameter

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was set to medium size. Other measurement conditions were as described above.

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Acquired mass spectra were normalized by total ion current (TIC) using FlexImaging 4.0

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software (Bruker Daltonics). The software was also used to create two-dimensional

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ion-density maps. The detection sensitivities were evaluated as the peak intensity

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variation of identified metabolite peaks by the two different matrix applications tested, i.e.,

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airbrush and TM-Sprayer, using the software.

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To compare the detection sensitivity of identified metabolites between the airbrush and

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TM-Sprayer methods, three sections prepared from the same strawberry fruit were

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analyzed. To investigate the spatial distributions of identified metabolites, three sections

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were prepared from four different strawberry fruits and analyzed. The representative ion

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images are shown.

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Statistical Analyses. The data are expressed as mean values with the standard 7 ACS Paragon Plus Environment


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deviation (SD). Significant differences between mean values were determined by

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Student’s t-test at the 5% significance level.

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RESULTS AND DISCUSSION

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Identification of Metabolites in Strawberry Extract. We performed MALDI-MS

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analysis to identify anthocyanins, sugars, and organic acids in ripe strawberry crude

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extract. Figure 1 shows the mass spectrum at m/z 200–600. First, to identify the

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anthocyanin species in the crude extract, precursor ions were analyzed by

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MALDI-MS/MS. In the MALDI- and DESI-MS/MS spectrum of the anthocyanin

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precursor ion, an intense peak of a molecular species was detected, which corresponded to

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anthocyanidin.20,21,28 The difference in m/z value between the peak of the precursor ion

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and the peak of anthocyanidin revealed the sugar moiety. The MS/MS spectrum of the m/z

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433.1 ion indicated an intense ion at m/z 271.0, which corresponded to the pelargonidin

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aglycone, and a neutral loss of 162.1 Da, which corresponded to the loss of a hexose

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moiety (Figure 2a). In several studies identifying anthocyanin species in strawberries,

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glucose rather than another hexose is known to be predominant at the hexose moiety of

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pelargonidin.10 Thus, we identified the m/z 433.1 ion as an [M]+ ion of

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pelargonidin-3-O-glucoside. Similarly, because the MS/MS spectra of the m/z 449.1 ion

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contained an intense ion at m/z 287.0 and its neutral loss was 162.1 Da (Figure 2b), the

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ion was identified as the [M]+ ion of cyanidin-3-O-glucoside.10 The MS/MS spectrum of

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the m/z 519.1, 535.1, and 579.1 ions contained intense ions at m/z 271.0, 287.0 and 271.0,

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and their neutral losses were 248.1, 248,1 and 308.1 Da (Figure 2d-f); therefore, these

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ions were also identified as the [M]+ ions of pelargonidin-3-O-rutinoside,

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cyanidin-3-O-rutinoside and pelargonidin-3-O-malonylglucoside, respectively, according 8 ACS Paragon Plus Environment

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to previous reports10. The MS/MS spectrum of the m/z 479.1 ions contained an intense ion

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at m/z 303.0, which corresponded to delphinidin (Figure 2c). However, the sugar moiety

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could not be identified from the neutral loss of 176.1. Thus, the m/z 479.1 ion was

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identified as an [M]+ ion of delphinidin-3-O-glycoside. Cyanidin and pelargonidin

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glycosides are commonly found in cultivated strawberries, but delphinidin-3-O-glycoside

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was identified for the first time in this study.

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The predominant sugars reported in strawberries are glucose, fructose, and sucrose,

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whereas the predominant organic acids are citric and malic acids.7–9 These were

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commonly detected as [M+Na]+ and/or [M+K]+ ions in plant tissues using MALDI- and

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DESI-IMS.22,23,28 In this study, hexoses (glucose and/or fructose), sucrose, and citric acid

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were predominantly detected as [M+K]+ ions at m/z 218.9, 380.9 and 230.9, respectively

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(Figure 1). The peak corresponding to malic acid was not observed. The anthocyanins,

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sugars, and organic acids identified in this study are summarized in Table 1.

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Comparison of Airbrush and Automatic Sprayer Methods. Preparation of thin

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tissue sections is crucial for successful MALDI-IMS.32,34 Generally, cutting thin slices of

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watery plant tissues such as those of many fruits is difficult due to crumbling and

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fracturing of the tissues.34 Thus, we compared a few preparation techniques for tissue

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sectioning. When frozen in liquid nitrogen and sectioned at 100-µm thickness, the

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maximum thickness setting on the cryostat used in this study, the external structures of

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strawberries, such as the skin, were destroyed. However, CMC freeze-embedding allowed

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us to obtain high-quality sections of whole strawberries with the structures remaining

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intact and all structures clearly observed when the section thickness was less than 100

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Matrix deposition is also crucial for successful MALDI-IMS.32,34 DHB solutions were

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sprayed using the airbrush and automatic sprayer on the strawberry sections before

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MALDI-IMS was performed. The ion images of the detected anthocyanins, sugars, and

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organic acid showed similar patterns between the airbrush and TM-Sprayer methods (data

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not shown). As shown in Figure 3, compared to the airbrush method, the automatic

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sprayer method provided higher average signal intensities of the identified metabolites.

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Furthermore, although the peak of delphinidin-3-O-glycoside at m/z 479.1 was not

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detected when using the airbrush, it was detected when using the automatic sprayer

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(Figure 3b). Similarly, it was previously reported that matrix application using the

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automatic matrix sprayer system resulted in approximately twice the number of

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metabolites detected, compared to the number detected when using the airbrush method.31

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The explanation for this effect is that the TM-Sprayer formed more homogeneous matrix

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crystals on the tissue than the airbrush.31,32 In addition, in the TM-sprayer method, DHB

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solution was sprayed at 85 °C, whereas, in the airbrush method, DHB solution was

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sprayed at room temperature. Thus, the improved signal intensities were also due to the

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higher extraction efficiency of metabolites from inside to the surface of tissues under the

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higher temperature of the DHB solution. Therefore, we used the TM-Sprayer for matrix

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spraying prior to distributional analysis of identified metabolites by MALDI-IMS.

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Distribution of Anthocyanins in Strawberries. The flesh of the strawberry fruit is a

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swollen receptacle, i.e., a false fruit, and the seeds or achenes located on the receptacle

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surface are the true fruit; these seeds are collectively referred to as the strawberry fruit.35

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To investigate the distributions of anthocyanins, sugars, and organic acids in ripe

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strawberry fruits, we performed MALDI-IMS analysis. Figure 4a shows the mass spectra 10 ACS Paragon Plus Environment

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detected from the strawberry section. All of the peaks corresponding to the metabolites

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identified in the crude extract (Figure 1) were also observed in the spectra from the

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sections obtained. Figure 4b is an optimal image of a longitudinal cross section of a

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strawberry. Red pigment is present in the skin, cortical and pith tissues of the fruit,

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indicating the presence of anthocyanins. First, we investigated the localization of

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anthocyanin molecular species. Interestingly, anthocyanin species showed different

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distribution patterns in the sections. Pelargonidin species were distributed in the skin,

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cortical and pith tissues (Figure 4d-f), whereas cyanidin and delphinidin species were

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localized in the skin (Figure 4h-i, k). In this study, pelargonidin aglycone, cyanidin

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aglycone and delphinidin aglycone were also detected (Figure 4c, g, j). These aglycones

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showed similar distribution patterns to their glycosides. These results suggested that the

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different distribution patterns were due to the difference in anthocyanin aglycone rather

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than to the sugar moieties. Anthocyanin distribution patterns in blueberry and black rice

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have been reported to be different. In the case of blueberries, the different distribution

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patterns of anthocyanins resulted from the difference in anthocyanin aglycones, rather

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than the sugar moieties,20 whereas in the case of black rice, the different distribution

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patterns of anthocyanins resulted from the different composition of sugar moieties, rather

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than the aglycones.21 These results suggest that the distribution patterns of anthocyanins

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are different among plant species, but that berry species have similar distribution patterns.

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Achenes are located on the receptacle surface and their color changes to red during

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ripening, indicating accumulation of anthocyanins. To investigate the distribution of

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anthocyanins in the achene, we prepared achene sections using CMC-embedding and

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performed MALDI-IMS analysis. Figure 5a shows an optical image of an achene section.

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As shown in Figure 5b-j, pelargonidin glycosides and cyanidin glycosides were detected, 11 ACS Paragon Plus Environment


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whereas delphinidin glycoside was slightly detected in the receptacle skin. The amounts

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of cyanidin glycosides were almost equal between the receptacle skin and the outside

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region of the achene skin, whereas those of pelargonidin glycosides were predominantly

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lower outside the achene skin than in the receptacle skin. These results suggest that the

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anthocyanin biosynthesis pattern was different between the receptacle skin and outside

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the achene skin.35

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The red color of strawberries is an important factor affecting consumer preference.

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The color of pelargonidin, cyanidin, and delphinidin-based anthocyanins is orange/brick,

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magenta/red, and blue/violet, respectively.36 These anthocyanins were localized in the

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receptacle skin, which indicated that the surface color is caused by the mixed balance of

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pelargonidin, cyanidin, and delphinidin, whereas the internal color is caused by

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pelargonidins. The pelargonidin glycosides and cyanidin glycosides are biosynthesized

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during the ripening period. It was observed that the color of ‘Tochiotome’ strawberries

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turns from red to dark red during late ripening. This might be because of an increase in

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delphinidin glycoside biosynthesis, in addition to an increase in pelargonidin glycoside

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and cyanidin glycoside biosynthesis.

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In the biosynthesis of anthocyanins in seed plants, pelargonidin, cyanidin and

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delphinidin glycosides are generated from the same precursor, dihydrokaempferol.36

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Dihydrokaempferol is converted to pelargonidin by the action of dihydroflavonol

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4-reductase (DFR), and by the subsequent action of anthocyanin synthase (ANS). In

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contrast, dihydrokaempferol is converted to dihydroquercetin and dihydromyricetin by

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the action of the flavonoid 3′-hydroxylase (F3′H) and the flavonoid 3′, 5′-hydroxylase

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(F3′5′H), respectively, and these compounds are further converted to cyanidin and

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delphinidin by the action of DFR and ANS, respectively. Thus, F3′H and F3′5′H are the 12 ACS Paragon Plus Environment

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key enzymes that regulate the balance of the biosynthesis of pelargonidin, cyanidin, and

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delphinidin.36 In this study, pelargonidin species were mainly distributed in the skin,

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cortical tissue, and pith tissue of the receptacle (Figure 4d-f), whereas cyanidin species

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were mainly localized in the receptacle skin and the outside of the achene skin (Figure

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4h-i, Figure 5g-h). Delphinidin species were slightly localized in the receptacle skin

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(Figure 4k). Compared to the detection intensity of pelargonidin aglycone, the detection

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intensities of cyanidin aglycone and delphinidin aglycone were about 16 and 175 times

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lower, respectively, in the receptacle skin, whereas that of cyanidin aglycone was about 9

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times higher in the outside of the achene skin, although it should be noted that this

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analysis was semi-quantitative.20,21 These results suggest that F3′H gene expression was

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limited and that the F3′5′H gene was only very slightly expressed in the skin of the

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receptacle, whereas F3′H gene was predominantly expressed in the outside of the achene

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skin. The biological significance of the different distribution patterns observed in

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anthocyanin species is currently not well understood. However, spatial information of

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anthocyanin species and their biosynthesis-related gene expression obtained by

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MALDI-IMS using different strawberry cultivars under various environmental conditions

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will contribute to understanding the biological significance in strawberries.20,21 It has been

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reported that overexpression of the anthocyanin synthase gene in strawberries enhances

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their antioxidant capacity and cytotoxic effect on human hepatic cancer cells2. Therefore,

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spatial information related to gene expression would assist those working in the field of

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breeding to enhance the prevention against chronic pathologies by improvement of the

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genetic properties of strawberries.

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Distribution of Sugars and Organic Acids in Strawberries. To investigate the 13 ACS Paragon Plus Environment


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localization of hexoses (glucose and/or fructose), sucrose, and citric acid, their ion images

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were constructed. As shown in Figure 4, hexoses were distributed over the entire

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strawberry section and the content was somewhat higher in the bottom side of the pith

292

than in any other tissue. Interestingly, sucrose was distributed predominantly in the top

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side of the cortical tissue and vascular bundle, and the content of sucrose was about 1.4 ±

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0.1 times higher in the top half than in the bottom half. It is well known that the top side is

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sweeter than the bottom side in the ripe ‘Tochiotome’ strawberry; therefore, in Japan, it is

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recommended to eat it from the bottom towards the tip. Multiple sugars and organic acids

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accumulate in ripe strawberry fruits.7–9 During fruit development, continuous import of

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sucrose from photosynthetic tissue is observed.35 Sucrose invertase activity is maintained

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high during all developmental stages of fruit, and imported sucrose is hydrolyzed by

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sucrose invertase into glucose and fructose. These three carbohydrates accumulate

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continually during strawberry fruit development, and are thus the major soluble sugars in

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the fruits.7,8 The detection intensity of sucrose in the strawberry sections was about 4.7 ±

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1.4 times higher than that of hexoses (Figure 4a), suggesting that sucrose content was

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higher than hexose content. The sweetness of glucose and fructose is 0.75 and 1.7 times

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that of sucrose, respectively.37 Therefore, it was suggested that one reason why the top

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side is sweeter than the bottom side of ripe ‘Tochiotome’ strawberries is the accumulation

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of sucrose in the top side of the pith and cortical tissues. This might be because of the

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increase in imported sucrose from photosynthetic tissue and/or decrease of invertase

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activity in the vascular bundle and top side of cortical tissues compared to other tissues of

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ripe strawberries.

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In this experiment, hexoses, such as glucose and fructose, could not be visualized

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separately due to the same m/z value using IMS and to the impossibility of detecting each 14 ACS Paragon Plus Environment

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specific fragment ion using IMS/MS. It is also necessary to investigate the distribution of

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glucose and fructose to estimate the sweetness of strawberries, because there is a

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possibility that glucose and fructose show different distribution patterns, as shown by the

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hexose image obtained in this study (Figure 4m). Recently, glucose and fructose were

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separately detected using ion mobility spectrometry-mass spectrometry analysis.13

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Therefore, it may be possible to visualize glucose and fructose separately using IMS

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equipped with ion mobility spectrometry.

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In conclusion, the major anthocyanins, sugars, and organic acids in ripe ‘Tochiotome’

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strawberry extract were identified using MALDI-MS. Longitudinal sections of whole

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strawberry fruits were prepared using CMC-embedding. The detection sensitivity of

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MALDI-IMS for identified metabolites in the fruit sections was improved by using the

324

TM-Sprayer for matrix application. MALDI-IMS analysis revealed the characteristic

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distribution patterns of identified metabolites in the fruits. The results obtained from this

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study may help breeders in their efforts to improve the quality of strawberry fruits.

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ABBREVIATIONS USED

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ANS, anthocyanin synthase; CMC, carboxymethyl cellulose; DESI, desorption

330

electrospray ionization; DFR, dihydroflavonol 4-reductase; DHB, 2,5-dihydroxybenzoic

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acid; F3’H, flavonoid 3’-hydroxylase; F3’5’H, flavonoid 3’, 5’-hydroxylase; IMS,

332

imaging mass spectrometry; LC, liquid chromatography; MALDI,

333

laser desorption ionization; MS/MS, tandem mass spectrometry; NL, neutral loss

matrix-assisted

334 335

ACKNOWLEDGMENTS

336

We thank Takayoshi Aoki for the helpful comments on our manuscript, and Ryosuke Sato 15 ACS Paragon Plus Environment


337

and Kotomi Matsumoto for the sample preparation. We gratefully acknowledge Kazuyuki

338

Koide and the Strawberry Research Center of the Tochigi Prefectural Agricultural

339

Experiment Station for providing ‘Tochiotome’ strawberries. We would also like to thank

340

Editage (www.editage.jp) for English language editing.

341 342

Funding

343

This work was supported by Japan Society for the Promotion of Science KAKENHI

344

(26292144) and by the Ministry of Education, Culture, Sports, Science, and

345

Technology-Supported Program for the Strategic Research Foundation at Private

346

Universities (2013–2017) [project number S131052A01].

347

Notes

348

The authors declare no conflict of interest.

349 350

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FIGURE CAPTIONS

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Figure 1. Mass spectrum obtained from strawberry crude extract using MALDI-MS.

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Figure 2. MS/MS spectra of identified anthocyanins in the strawberry crude extract using

484

MALDI-MS/MS. Representative MS/MS spectra of precursor ions at m/z (a) 433.1, (b)

485

449.1, (c) 479.1, (d) 519.1, (e) 535.1, and (f) 579.1. Neutral losses (NL) indicate the

486

losses of sugar moieties.

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Figure 3. Comparison of detection sensitivities of anthocyanins, organic acids, and sugars

488

in the different three strawberry sections using airbrush or TM-Sprayer for matrix

489

application. Data represent the mean ± SE of detection intensities at (a) m/z 433.1, 519.1,

490

579.1, (b) 449.1, 535.1, 479.1, (c) 230.9, 219.0, and 381.0 (n=3). ∗ indicates significant

491

differences compared with airbrush (p < 0.05) for each metabolite as determined by

492

Student’s t-test. n.d. indicates not detected.

493

Figure 4. MALDI-IMS analysis of anthocyanins, sugars, and organic acids in strawberry

494

sections. (a) Mass spectrum obtained from a section. (b) Optical image of a strawberry

495

section. The dotted line shows the analyzed region. (c-n) Representative ion images of

496

m/z 271.0, 433.1, 519.1, 579.1, 287.0, 449.1, 535.1, 303.0, 479.1, 219.0, 381.0, and

497

230.9. Scale bar = 5 mm.

498

Figure 5. MALDI-IMS analysis of anthocyanins in an achene. (a) Optical image of an

499

achene section. All of the region shown by the image was analyzed. (b-j) Representative

500

ion images of m/z 271.0, 433.1, 519.1, 579.1, 287.0, 449.1, 535.1, 303.0, and 479.1.

501

Scale bar = 1 mm.

502 503 504 22 ACS Paragon Plus Environment

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Table 1. Anthocyanins, Sugars and Organic Acids Identified in this Study.

group

parent ion

fragment ion

(m/z)

(m/z)

identified metabolites

[M]+

[M]+

433.1

271.0

pelargonidin-3-O-glucoside

449.1

287.0

cyanidin-3-O-glucoside

479.1

303.0

delphinidin-3-O-glycoside

519.1

271.0

pelargonidin-3-O-malonylglucoside

535.1

287.0

cyanidin-3-O-malonylglucoside

579.1

271.0

pelargonidin-3-O-rutinoside

[M+K]+

[M+K]+

219.0

n.d.a

381.0

201.0, 219.0

230.9

n.d.a

anthocyanin

hexose (glucose and/or fructose)

sugar organic acid a

sucrose citric acid

n.d., not detected.



Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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TOC Graphic


Distribution analysis of anthocyanins, sugars, and organic acids in

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