Anal Bioanal Chem (2012) 403:1885–1895 DOI 10.1007/s00216-012-5876-z

ORIGINAL PAPER

Visualization of anthocyanin species in rabbiteye blueberry Vaccinium ashei by matrix-assisted laser desorption/ionization imaging mass spectrometry Yukihiro Yoshimura & Hirofumi Enomoto & Tatsuya Moriyama & Yukio Kawamura & Mitsutoshi Setou & Nobuhiro Zaima

Received: 29 November 2011 / Revised: 12 February 2012 / Accepted: 14 February 2012 / Published online: 8 March 2012 # Springer-Verlag 2012

Abstract Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants, and are believed to have a number of beneficial health effects in both humans and animals. Because of these properties, pharmacokinetic analysis of anthocyanins in tissue has been performed to quantify and identify anthocyanin species although, currently, no methods exist for investigating tissue localization of anthocyanin species or for elucidating the mechanisms of anthocyanin activity. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of a wide range of biomolecules. To investigate whether anthocyanin species could be identified and visualized by IMS, we performed matrix-assisted laser desorption/ionization (MALDI)–IMS analysis, by tandem mass spectrometry (MALDI–IMS–MS), of ten anthocyanin molecular species in rabbiteye blueberry (Vaccinium ashei).

Published in the special paper collection Biomedical Mass Spectrometry with guest editors Toyofumi Nakanishi and Mitsutoshi Setou. Y. Yoshimura : T. Moriyama : Y. Kawamura : N. Zaima (*) Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, 3327-204 Naka-machi, Nara 631-8505, Japan e-mail: [email protected] H. Enomoto Department of Biosciences, Faculty of Science and Engineering, Teikyo University, Utsunomiya, Tochigi 320-8551, Japan M. Setou Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan

The distribution patterns of each anthocyanin species were different in the exocarp and endocarp of blueberry sections. Anthocyanin species composed of delphinidin and petunidin were localized mainly in the exocarp. In contrast, those species composed of cyanidin, peonidin, and malvidin were localized in both the exocarp and the endocarp. Moreover, MALDI–IMS analysis of anthocyanidins in a blueberry section indicated that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins. These results suggested that the different distribution patterns of anthocyanin species in the exocarp and endocarp depended on the aglycone rather than on the sugar moieties. This study is the first to visualize anthocyanin molecular species in fruits. Keywords Agriculture . Foods/beverages . Mass spectrometry/ICP–MS . Natural products

Introduction Anthocyanins are ubiquitously occurring polyphenols in the plant kingdom that impart a variety of red–blue colors to fruits, vegetables, and plants. They belong to the widespread class of phenolic compounds collectively named flavonoids. They are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium (Fig. 1a). Common species of anthocyanidins observed in higher plants are classified according to the number and position of the hydroxyl groups on the flavan nucleus, and include cyanidin, delphinidin, malvidin, peonidin, pelargonidin, and petunidin (Fig. 1b). It has been suggested that more than 400 anthocyanins have already been identified in nature [1], where their ability to impart color to the plants or plant products attracts

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Visualization of anthocyanin species in rabbiteye blueberry

R

Fig. 1 Structures of anthocyanins and the general flavonoid biosynthesis pathway leading to anthocyanins. a Structures of anthocyanins. Common sugar moieties found in the blueberry anthocyanins are arabinose, glucose, and galactose. b Structures of common anthocyanidins found in higher plants. R1 and R2 denote the positions shown in the anthocyanin structure (a). c General flavonoid biosynthetic pathway leading to anthocyanins found in blueberries. Key biosynthetic enzymes are shown in boldface. 3GT, 3-O-glycosyltransferase; ANS, anthocyanidin synthase; AOMT, anthocyanin O-methyltransferase; DFR, dihydroflavonol 4-reductase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; Gly, glycoside

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blueberry fruits as samples, because blueberry is rich in anthocyanins [27, 28] and has recently been focused on because of numerous reports of its beneficial effects on human health [5, 29–31]. In this study we identified and visualized ten species of anthocyanin and indicated the different distribution patterns of each species in rabbiteye blueberry (Vaccinium ashei) by MALDI–IMS. This work is the first visualization of anthocyanin molecular species in fruit.

Materials and methods animals for pollination and seed dispersal. Recently, much interest in anthocyanins has emerged not only because of their functions in plants but also other properties, including potential applications as anti-diabetics [2], anti-cancer drugs [3] and antinociceptives [4]. Anthocyanins may also protect against cardiovascular damage [5], amyloid β-peptide-induced degeneration [6], and light-induced retinal damage [7] because of their antioxidant and anti-inflammatory properties [8]. Despite these potential benefits of anthocyanins, little is known about their potential harmful effects, although no evidence of toxicity has yet been reported, despite widespread consumption of food products that contain anthocyanins [9]. Several studies have indicated that anthocyanins are absorbed from the intestinal tracts of humans and animals and excreted in the urine or feces [10–12]. In some animal models, anthocyanins have been detected in the liver, kidney, eye, and brain after supplementation, indicating they are able to cross the blood– brain barrier [13–15]. Studies have reported rapid detection of anthocyanins in different tissues—the compounds were detected in the brain less than 30 min after oral administration [13] and in the kidney and liver 15 s after intravenous administration [16]. These results suggest that anthocyanins are rapidly taken up from the blood into tissues, where they accumulate up to their bioactivity threshold. Such pharmacokinetic analysis of anthocyanins in tissue has been performed primarily by using LC–MS to quantify and identify the anthocyanin species; however, any information related to local distribution and concentration of each anthocyanin species in the tissue is completely lost as a result of the destructive nature of these experiments. This information is very important if we are to elucidate the mechanisms of action of anthocyanins; to date, however, methods for investigating anthocyanin localization, for example determining specific antibodies to each anthocyanin species, have not yet been developed. MALDI–IMS is an emerging technique that enables simultaneous investigation of both the content and spatial distribution of a wide range of biomolecules, for example lipids [17–19], glycolipids [20, 21], amino acids [22], proteins [23], peptides [24, 25], and administered pharmaceuticals [26], without requiring antibodies, staining, or complicated pretreatment steps. To establish a method for investigation of the distribution patterns of each anthocyanin species by IMS, we chose

Materials Glass slides (Fisher brand Superfrost Plus) were purchased from Thermo Fisher Scientific (San Jose, CA, USA). Methanol and distilled water were purchased from Nacalai Tesque (Kyoto, Japan). 2,5-Dihydroxybenzoic acid (DHB) was obtained from Bruker Daltonics (Bremen, Germany). All chemicals used in this study were of the highest purity available. Rabbiteye blueberry (Vaccinium ashei) cultivars Representative samples of blueberries were collected at random from the cultivars Whitu, Rahi, Brightwell, Premier, and Powderblue, which were harvested in Nara, Japan. Anthocyanin extraction from rabbiteye blueberries Fresh blueberries were dried by use of an evaporator at room temperature and then delipidated by treatment with hexane and ethanol to eliminate interference from lipid components. Anthocyanins were extracted from a delipidated blueberry sample by use of 50% ethanol at room temperature for 2 h. The crude extract was analyzed by MALDI–mass spectrometry (MALDI–MS) and MALDI–tandem mass spectrometry (MALDI–MS–MS) to identify the anthocyanin species. MALDI–MS and MS–MS analysis of anthocyanins in crude blueberry extract Crude blueberry extract (5 μL) was mixed with an equal volume of 50 mg mL−1 DHB in methanol–water 7:3 (v/v). The mixed sample was deposited on to a glass slide and dried before MALDI–MS analysis. MALDI–MS analysis was performed using a MALDI linear quadrupole ion-trap mass spectrometer (MALDI LTQ-XL; Thermo Fisher Scientific) equipped with a 337-nm nitrogen laser used at a repetition rate of 60 Hz in positive-ion mode. The laser energy was set to 20 μJ, and the mass spectrometer was operated in automatic gain control (AGC) mode. Ions with m/z values in the range 400–650 were measured. For MS–

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MS analysis, the selected precursor ions and the product ions obtained by collision-induced dissociation were ejected from the ion trap and analyzed. The collision energy was set to 18% of the maximum available energy required to completely fragment the precursor ion formed from the peptide Met–Arg–Phe–Ala [32]. Preparation of cryosections Rabbiteye blueberries were frozen in liquid nitrogen and stored at −65°C. The frozen blueberries were sliced to 50-μm thickness using a cryostat (CM 1850; Leica Microsystems, Wetzler, Germany), generating cross sections. The sections were thaw-mounted on glass slides and then air-dried immediately. Images of the sections were obtained by use of a 3R Systems (Japan) 3R-MSA200 digital mobile microscope before IMS analysis. Imaging mass spectrometry MALDI–IMS analysis was performed using the LTQ-XL mass spectrometer as described above. Briefly, 50 mg mL−1 DHB in methanol–water 7:3 (v/v) was used as matrix. The DHB matrix solution (500 μL) was sprayed uniformly over the blueberry sections by use of an airbrush with a 0.2-mm nozzle (Procon Boy FWA Platinum; Mr Hobby, Tokyo, Japan). Data were acquired with a 100-μm step size in positive-ion mode. The laser energy was set to 20 μJ, and the mass spectrometer was operated in AGC mode. Ions with m/z values in the range of 150–1000 were measured. ImageQuest software (Thermo Fisher Scientific) was used to create two-dimensional ion-density maps, normalize peak intensity, adjust the color scale, and quantify the ion intensity. For MALDI–IMS based on MS–MS (MALDI–IMS– MS) analysis of the anthocyanins in the sections, the collision energy was set to 18% of the maximum available energy for the LTQ-XL, and the laser energy was set to 20 μJ.

Results and discussion Identification of anthocyanin species in the crude extract of rabbiteye blueberries Anthocyanins are usually extracted from plant materials by use of methanol acidified with small amounts of hydrochloric acid or formic acid [33, 34]. Although these methods have been shown to be effective for anthocyanin extraction, it has been suggested that even small amounts of acid may cause partial hydrolysis of anthocyanins [1]. In addition to acidified methanol, aqueous ethanol is also an effective solvent for extraction of anthocyanins from plant materials

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such as dried blueberries and black rice seeds [7, 35]; therefore, we extracted anthocyanins from blueberries with 50% ethanol to prevent degradation of anthocyanins. We then performed MALDI–MS analysis to identify the anthocyanins in the crude extract from rabbiteye blueberries. As shown in Fig. 2a, abundant anthocyanin signals at m/z 419, 433, 435, 449, 463, 465, 479, and 493 were observed in the mass spectrum. We analyzed precursor ions by MALDI– MS–MS to identify the molecular species of anthocyanins in the crude extract. The MS–MS spectrum of the m/z 419 ion in the crude extract indicated an intense ion at m/z 287 (neutral loss of 132 Da), which would correspond to loss of a pentose moiety (Fig. 2b). The MS–MS fragmentation of the ion at m/z 287 would correspond to the cyanidin aglycone. We could not identify the sugar moiety of this anthocyanin species because the MALDI–MS system could not differentiate between molecular species of sugar moieties that have the same molecular mass, for example xylose and arabinose. Several studies on identification of anthocyanin species by liquid chromatography have shown that the pentose moiety of anthocyanins from rabbiteye blueberries (Vaccinium ashei) or other blueberry species is predominantly an arabinose rather than another pentose such as xylose [34–36]. Thus, we identified the m/z 419 ion as an [M]+ ion of cyanidin-3-O-arabinoside (Fig. 2b). Similarly, because the ions in the blueberry crude extract at m/z 433 and 435 also yielded the characteristic neutral loss of 132 Da corresponding to the loss of an arabinose moiety, the ions were identified as the [M]+ ions of peonidin-3-O-arabinoside (Fig. 2c) and delphinidin-3-O-arabinoside (Fig. 2d), respectively. The MS–MS spectrum of the m/z 449 ion in the crude extract contained intense ions at m/z 287 (neutral loss of 162 Da) and 317 (neutral loss of 132 Da), which would correspond to loss of a hexose moiety and an arabinose moiety, respectively (Fig. 2e). Because the hexose moieties of anthocyanins from blueberries contain both galactose and glucose [34–36], we could not differentiate between molecular species of hexose moieties having the same molecular mass. The fragmentation ions at m/z 287 and 317 derived from the precursor ion would correspond to the cyanidin and petunidin aglycones, respectively. Therefore, the precursor ions at m/z 449 contain ions from three anthocyanin species, cyanidin-3-O-hexoside (cyanidin-3-Ogalactoside or cyanidin-3-O-glucoside), and petunidin-3-Oarabinoside (Fig. 2e). Similarly, the precursor ions at m/z 463 contain ions from peonidin-3-O-hexoside (peonidin-3O-galactoside or peonidin-3-O-glucoside) and malvidin-3O-arabinoside (Fig. 2f). The molecular species identified in MS–MS analyses are summarized in Table 1. Similar to previous reports, the anthocyanin species containing pelargonidin as aglycone could not be detected in our experiments. Our results from MS analysis of rabbiteye blueberry anthocyanins revealed a peak at m/z 493 corresponding to

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Fig. 2 Identification of anthocyanin species in rabbiteye blueberry crude extract. a Mass spectrum obtained from rabbiteye blueberry crude extract. b–f Representative MS–MS spectra of ions in rabbiteye

blueberry crude extract, [M]+ ions at m/z (b) 419, (c) 433, (d) 435, (e) 449, and (f) 463. Neutral losses (NL) of 132 Da and 162 Da indicate losses of an arabinose and hexose, respectively

the [M]+ ion of malvidin-3-O-hexoside as the primary peak (Fig. 2a). This result is consistent with data from a previous study on the identification and quantification of anthocyanins in rabbiteye blueberries that showed malvidin-3-Ogalactoside was the primary anthocyanin species [36]. Recent studies have shown that anthocyanins derived from blueberries were absorbed and detected in human plasma and urine after consumption of blueberries [10,

11]. Moreover, blueberry and other berry anthocyanins ingested by rats could have been absorbed and delivered to the liver and eyes, eventually crossing the blood–brain barrier [13–15, 37], suggesting that anthocyanins can feasibly have a direct effect on brain processes and the function of other organs. In cultured neuron cells, neuro-2A, malvidin aglycone, and malvidin-3-O-glucoside, which are abundant in blueberries (Fig. 2a), had protective effects against

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Table 1 Anthocyanin species identified in this study +

Parent ion [M]+ (m/z)

Fragment ion [M] (m/z)

Anthocyanin identified

419

287

Cyanidin-3-O-arabinoside

433

301

Peonidin-3-O-arabinoside

435 449

303 287

Delphinidin-3-O-arabinoside Cyanidin-3-O-hexoside

449

317

Petunidin-3-O-arabinoside

463 463

301 331

Peonidin-3-O-hexoside Malvidin-3-O-arabinoside

465

303

Delphinidin-3-O-hexoside

479 493

317 331

Petunidin-3-O-hexoside Malvidin-3-O-hexoside

amyloid β-induced neurotoxicity by blocking the formation of reactive oxygen species [6]. Compared with malvidin-3O-hexoside, cyanidin-3-O-glucoside is not a primary anthocyanin species in blueberries (Fig. 2a, e), but it is the most common anthocyanin in higher plants [1]. Cyanidin-3-Oglucoside has been recognized as a potent bioactive molecule that protects against TNF-α-induced endothelial dysfunction [38], ethanol neurotoxicity in the developing brain [39], and tumor promoter-induced carcinogenesis and tumor metastasis [40]. Malvidin aglycone and malvidin-3-O-glucoside, also, have similar effects, which may be related to high antioxidative activity. Recently, it was reported that small hydrophobic molecules derived from plants containing anthocyanin aglycone, anthocyanidin, inhibited neurotransmitter release from neuronal PC12 cells by inhibiting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation [41]. Interestingly, cyanidin and delphinidin, but not malvidin, have been found to be potent inhibitors of SNARE complex formation, which indicates that each anthocyanin or anthocyanidin molecular species has its own functions or bioactivity in organisms. Therefore, investigating the functions of the individual anthocyanin species is essential.

MALDI–IMS analysis of anthocyanins in rabbiteye blueberry sections Investigating the distribution of each anthocyanin molecular species in blueberries will not only be useful for elucidating the effects of blueberry consumption on human health, but also for determining the biological functions of each anthocyanin molecular species. Figure 3a is an image of a crosssection of a rabbiteye blueberry. Purple pigment is present in both the exocarp and the endocarp of the cultivar. First, we performed MALDI–IMS analysis on the blueberry section to investigate the types and locations of anthocyanin

molecular species. Figures 3b–e show mass spectra obtained from the each part of the section, i.e. the outer region of blueberry tissue (matrix), exocarp, mesocarp, and endocarp, respectively. All the peaks corresponding to the anthocyanins observed in the crude anthocyanin extract (Fig. 2b) were also identified in the spectra from the exocarp and endocarp (Fig. 3c, e). Peaks observed at m/z 433 and 435 in the spectrum obtained from the mesocarp (Fig. 3d) are believed to arise from peonidin-3-O-arabinoside and delphinidin-3-O-arabinoside, respectively. Because these peaks were also observed in the mass spectrum obtained from the matrix region (Fig. 3b), and because the purple pigment is not present in the mesocarp (Fig. 3a), the ions corresponding to the peaks observed in the mass spectra were partially derived from the matrix. In fact, the ions at m/z 433 and 455 were distributed in not only blueberry tissues but also in the matrix region of the MALDI–IMS image (Fig. 3f, data not shown). Although other ions corresponding to anthocyanins were distributed in the exocarp and endocarp (Fig. 3g, h, data not shown) in the MALDI–IMS image, the ions at m/z 449 and 463 contained the distribution patterns of at least two species of anthocyanin, as determined by MS–MS analysis of the crude extract (Fig. 2e, f). Moreover, it is possible that the distribution patterns of all the ions at m/z 419, 433, 435, 449, 463, 465, 479, and 493 contain peaks from other ions with the same m/z value, and therefore could not be differentiated by MS. Therefore, MALDI–IMS analysis is thought to be unsuitable for investigation of the distribution pattern of each anthocyanin molecular species. MALDI–IMS based on MS–MS analysis of anthocyanins in rabbiteye blueberry sections We subsequently performed MALDI–IMS–MS on the blueberry sections to investigate the local distribution of each anthocyanin species. We identified ten anthocyanin species in the exocarp and endocarp of blueberry as well as in the crude extract (Fig. 4n). MALDI–IMS–MS analysis was used to investigate the distribution patterns of peonidin-3O-arabinoside (m/z 433→301) and delphinidin-3-O-arabinoside (m/z 435→303), which could not be investigated adequately by MALDI–IMS analysis (Fig. 4d, g). Moreover, MALDI–IMS–MS enabled us to differentiate between anthocyanin species having the same m/z value as precursor ions, for example the ions at m/z 449 (cyanidin-3-O-hexoside (m/z 449→287, Fig. 4c) and petunidin-3-O-arabinoside (m/z 449→317, Fig. 4i)) and 463 (peonidin-3-O-hexoside (m/z 463→301, Fig. 4e) and malvidin-3-O-arabinoside (m/z 463→331, Fig. 4l)). Interestingly, the distribution patterns of each anthocyanin species were different in the exocarp and endocarp. Cyanidin-3-O-arabinoside (m/z 419→287, Fig. 4b), cyanidin-3-O-hexoside (m/z 449→287, Fig. 4c),

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Fig. 3 MALDI–IMS analysis of anthocyanins in a rabbiteye blueberry section. a Optical image of the rabbiteye blueberry cross-section used in the following MALDI–IMS analyses (b–h). b–e The mass spectra obtained from each part of the section corresponding to: b the matrix, the outer region of blueberry tissue, c exocarp, d mesocarp, and e endocarp. f– h MALDI–IMS images of the ions at m/z (f) 433, (g) 449, and (h) 493. Scale bar: 2.0 mm

peonidin-3-O-arabinoside (m/z 433→301, Fig. 4d), and peonidin-3-O-hexoside (m/z 463→301, Fig. 4e) were localized almost equally in the exocarp and endocarp. Delphinidin-3-O-arabinoside (m/z 435→303, Fig. 4g), delphinidin-3-O-hexoside (m/z 465→303, Fig. 4h), petunidin-3-O-arabinoside (m/z 449→317, Fig. 4i), and petunidin-3-O-hexoside (m/z 479→317, Fig. 4j) were less evident in the endocarp. As shown in Fig. 4f, the purple pigment was abundant in the endocarp of the section; thus, the lower abundance of these anthocyanin species in the

endocarp was not because of lack of endocarp pigment in the sections. However, malvidin-3-O-arabinoside (m/z 463→331, Fig. 4l) and malvidin-3-O-hexoside (m/z 493→331, Fig. 4m) were most abundant in the exocarp. These results suggest that the different distribution patterns of anthocyanin species in the exocarp and endocarp were because of different compositions of aglycone rather than because of the sugar moieties. Recently, we also investigated the localization of anthocyanin species in black rice and found that anthocyanin species containing a pentose moiety

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Fig. 4 MALDI–IMS–MS analysis of anthocyanins in a rabbiteye blueberry section. a Optical image of the blueberry cross-section used in MALDI–IMS–MS analyses (b–e). b–e MALDI–IMS–MS images of the fragment ions derived from the precursor ions of (b) cyanidin-3-Oarabinoside, (c) cyanidin-3-O-hexoside, (d) peonidin-3-O-arabinoside, and (e) peonidin-3-O-hexoside. f Optical image of the blueberry crosssection used in MALDI–IMS–MS analyses (g–j). g–j MALDI–IMS– MS images of the fragment ions derived from the precursor ions of (g)

delphinidin-3-O-arabinoside, (h) delphinidin-3-O-hexoside, (i) petunidin-3-O-arabinoside, and (j) petunidin-3-O-hexoside. k Optical image of the blueberry cross-section used in MALDI–IMS–MS analyses (l, m). l, m MALDI–IMS–MS images of the fragment ions derived from the precursor ions of (l) malvidin-3-O-arabinoside, and (m) malvidin-3-O-hexoside. n Summary of MALDI–IMS–MS analysis of the blueberry sections. Scale bar: 2.0 mm

(cyanidin-3-O-pentoside and petunidin-3-O-pentoside) were localized in the entire pericarp, whereas anthocyanin species containing a hexose moiety (cyanidin-3-Ohexoside and peonidin-3-O-hexoside) were focally localized in the dorsal pericarp [42]. Different from blueberry, the different distribution patterns of anthocyanin species in black rice were due to the different compositions of sugar moieties rather than due to the aglycones. The patterns of the anthocyanins in these two plants suggests that biological species-specific mechanisms underlie the different distributions of anthocyanin molecular species.

301, Fig. 5c) were localized almost equally at the exocarp and endocarp, whereas only trace amounts of delphinidin (m/z 303, Fig. 5d) and petunidin (m/z 317, Fig. 5e) were found in the endocarp. Malvidin (m/z 331, Fig. 5f) was present in both the exocarp and endocarp, and was abundant in the exocarp. Figure 5g shows results from semiquantitative analysis of anthocyanidin species in the exocarp and endocarp of blueberry sections. In the exocarp, malvidin was the major species; the ion intensity of the others, including cyanidin, peonidin, delphinidin, and petunidin, was one-fifth that of malvidin. In the endocarp, the ion intensity of malvidin was less than that in the exocarp, but was higher than that of the other anthocyanidin species. The ion intensities of cyanidin and peonidin in the endocarp were equal to those in the exocarp. However, the ion intensities of delphinidin and petunidin were lower in the endocarp than in the exocarp. These result indicate that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins (Fig. 4). The MALDI–IMS analysis of anthocyanidins in blueberry sections supported the hypothesis that the different distribution patterns of anthocyanin species in the exocarp and endocarp

MALDI–IMS analysis of anthocyanidins in rabbiteye blueberry sections To confirm the distribution patterns of anthocyanins, the aglycones of anthocyanin, we investigated the distribution patterns of anthocyanidins by MALDI–IMS analysis. In this experiment, anthocyanidin species could be detected simultaneously and semi-quantitatively in the same blueberry section. Cyanidin (m/z 287, Fig. 5b) and peonidin (m/z

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Fig. 5 MALDI–IMS analysis of anthocyanidins, the aglycones of anthocyanin, in a rabbiteye blueberry section. a Optical image of the rabbiteye blueberry cross-section used in the following MALDI–IMS analyses (b–g). b–f MALDI– IMS images of the ions at m/z (b) 287, (c) 301, (d) 303, (e) 317, and (f) 331 corresponding to cyanidin, peonidin, delphinidin, petunidin, and malvidin, respectively. g Semiquantification of the ion intensities of each anthocyanidin in the exocarp and endocarp. Results were collected from three sections, and error bars represent mean ± SD. Scale bar: 2.0 mm

were due to different aglycone compositions rather than due to sugar moieties. These results, however, did not explain the cause of the distribution patterns of each anthocyanin species in blueberries. In the anthocyanin biosynthesis pathway in several plants, pelargonidin, cyanidin, and delphinidin are generated first from a flavone, naringenin, via several reactions (Fig. 1c) [43]. These anthocyanidin species are further converted into anthocyanins by addition of a sugar moiety. Furthermore, cyanidin/cyanidin-3-O-glycoside and delphinidin/delphinidin-3-O-glycoside are methylated at the 3′ position by anthocyanin O-methyltranferases (AOMT) to generate peonidin/peonidin-3-O-glycoside and petunidin/ petunidin-3-O-glycoside, respectively. Malvidin/malvidin3-O-glycoside is generated by methylation at the 3′ position of petunidin/petunidin-3-O-glycoside or at the 3′ and 5′ positions of delphinidin/delphinidin-3-O-glycoside (Fig. 1c). Recently, cation-dependent AOMT which were

able to methylate at both the 3′ and 5′ positions of anthocyanin molecule were identified in grapevines [44, 45]. Although the methyltransferase genes which are responsible for methylation of anthocyanins have not yet been identified in blueberries, the 3′,5′-O-methyltransferase could act effectively in the exocarp and endocarp in blueberries, because anthocyanins composed of malvidin, a highly methylated anthocyanidin, are the most abundant in blueberries and in grapevines which contain AMOTs [46]. Nonmethylated anthocyanidins, cyanidin and delphinidin, are generated from the same precursor, dihydrokaempferol (Fig. 1c). Dihydrokaempferol is converted to dihydroquercetin and dihydromyricetin by the action of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), respectively. Dihydroquercetin and dihydromyricetin are further converted by dihydroflavonol 4-reductase (DFR) to leucocyanidin and leucodelphinidin, respectively. Finally, leucocyanidin and leucodelphinidin are converted to

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cyanidin and delphinidin, respectively (Fig. 1c), by the action of anthocyanin synthase (ANS). Thus, F3′H and F3′ 5′H are the key enzymes that determine the balance of cyanidin/peonidin and delphinidin/petunidin/malvidin, and AOMTs are the enzymes that determine the amounts of methyl groups at the 3′ and 5′ positions of cyanidin and delphinidin. Results from our semi-quantitative analysis of anthocyanidins are summarized in Fig. 5g; MALDI–IMS analysis showed that cyanidin (Fig. 5b) and its monomethylated product, peonidin (Fig. 5c), were localized equally in the exocarp and endocarp, whereas delphinidin (Fig. 5d) and its methylated products, petunidin (Fig. 5e) and malvidin (Fig. 5f), were more abundant in the exocarp than in the endocarp, suggesting that the F3′H gene was expressed at the same level in the exocarp and endocarp, and that F3′5′H gene expression was higher in the exocarp than in the endocarp. In addition, it has recently been reported that expression of the F3′H and F3′5′H genes was elevated during fruit ripening when the anthocyanins were accumulated in a cultivar of highbush blueberry (Vaccinium corymbosum L.) [47] suggesting that enzymatic activities of F3'H and F3'5'H might exist in the cultivars used in this study. Although the reasons for and biological significance of the different distribution patterns of each anthocyanin species are currently not well understood, such analyses of gene expression related to anthocyanin biosynthesis and information obtained by MALDI–IMS and IMS–MS about the distribution pattern of each anthocyanin/anthocyanidin molecular species in different blueberry cultivars will improve our understanding of the biological significance of anthocyanin species in blueberry, and assist scientists working in the field of breeding to improve the genetic properties of blueberries.

Conclusions We identified and visualized the anthocyanin species in rabbiteye blueberry sections with MALDI–IMS–MS and found that the distribution patterns of each anthocyanin species were different in the exocarp and endocarp. We also found that MALDI–IMS analysis using DHB as matrix was not suitable for investigating the distribution patterns of anthocyanin species, because some ions derived from the matrix solution had the same m/z values as the ions corresponding to peonidin-3O-arabinoside and delphinidin-3-O-arabinoside, and because MALDI–IMS is not able to differentiate between anthocyanin species having the same m/z values, for example cyanidin-3O-hexoside and petunidin-3-O-arabinoside at m/z 449. Results of this study will contribute to better understanding of the biological significance of anthocyanin species in blueberries, and to pharmacokinetic analysis of the post-consumption tissue localization of anthocyanin species, for elucidating the mechanisms of action of each anthocyanin species.

Y. Yoshimura et al. Acknowledgements This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Biooriented Industry (BRAIN) to N.Z.

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