Distribution of Flavan-3-ol Species in Ripe Strawberry Fruit Revealed by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging

Flavan-3-ols, which comprise proanthocyanidins and their monomers, are major flavonoids in strawberries, and they have a wide range of biological activities and health benefits. However, their spatial distribution in strawberry fruit remains poorly understood. Therefore, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), to visualize flavan-3-ols in ripe strawberry fruit. Peaks matching the m/z values of flavan-3-ols [M − H]− ions were detected in the negative ion mode using 1,5-diaminonaphthalene as matrix. Catechin and/or epicatechin, three B-type procyanidins, and two B-type propelargonidins were identified by MALDI-tandem MS. These flavan-3-ols were mainly distributed in the calyx, in and around the vascular bundles, and in the skin. In-source fragmentation of proanthocyanidins was determined using their standards, suggesting their distribution was mixed ion images of themselves, and fragment ions generated from those had a higher degree of polymerization. B-type procyanidins were predominantly distributed in the vascular bundles than in the skin, whereas B-type propelargonidins were almost equally distributed between the vascular bundles and skin, suggesting that their distribution patterns are different from the type of their flavan-3-ol monomers. Flavan-3-ols, especially B-type procyanidins, may help prevent pathogen infection not only in the skin but also in and around the vascular bundles.


Introduction
Proanthocyanidins, also known as condensed tannins, are oligomers and polymers of flavan-3-ol units that have been used as therapeutic compounds since ancient times [1,2]. Flavan-3-ols are one of the most ubiquitous groups of plant phenolics; they are synthesized via the phenylpropanoid and flavonoid pathways [1][2][3] and are distributed widely in plants. Flavan-3-ols also have diverse biological activities, including protection against herbivorous animals and pathogen attacks (both bacterial and fungal), and are effective in restricting the growth of neighboring plants [1]. Flavan-3-ols are also widely distributed in human food of plant origin, particularly fruits, legume seeds, and cereal grains; where, they contribute to the bitter flavor and astringency [1]. In recent years, considerable attention Strawberries (Fragaria × ananassa) are the most widespread and consumed fruits in the world owing to their attractive appearance, taste, and health-related properties [10,11]. Strawberries are rich in nutritious compounds, including, vitamins, and organic acids, as well as non-nutritious phytochemicals, especially polyphenols [10,11]. Data from several studies using strawberry fruit powder or extracts suggested that these nutritious compounds promote beneficial health effects, such as preventative functions against cancer [10][11][12][13][14]. Flavan-3-ols, namely proanthocyanidins and their monomers, are the second-most abundant polyphenols after anthocyanins in strawberry fruit [11]. Thus, in addition to other classes of metabolites, flavan-3-ols are also believed to be associated with the health properties of strawberry fruit. However, the spatial distributions of flavan-3-ols in the bodies of strawberry fruit have not been fully determined.
In this study, using ripe 'Tochiotome' strawberries (a popular strawberry in Japan), we identified B-type propelargonidin and procyanidin species using MALDI-MS/MS analysis. Subsequently, we visualized the spatial distributions of the identified proanthocyanidins and showed their unique distribution by MALDI-MSI analysis. We expect that these results will contribute to further elucidating the biological roles of flavan-3-ol species.

Mass Spectra Obtained from Strawberry Fruit Sections
It has been reported that several proanthocyanidins, namely B-type procyanidin and propelargonidin oligomers, and their flavan-3-ol monomers are present in strawberry fruit [31,32]. Next, to investigate whether proanthocyanidins and their monomers can be detected in strawberry fruit sections, we prepared the fruit sections using carboxymethyl cellulose (CMC)-freeze embedding [24] and performed MALDI-MSI analyses in the negative-ion mode, using DAN as a matrix. Figure 2A shows the optical image of strawberry section before MALDI-MSI. Five tissues, namely the skin, cortical tissue, vascular bundles, pith tissue, and calyx were observed. Figure 2B Figure 2C-I, all potential components of flavan-3-ols were mainly distributed in the calyx, in and around the vascular bundles, and in the skin.   Typical fragmentation patterns of B-type proanthocyanidins observed by liquid chromatography (LC)-MS/MS or MALDI-MS/MS following RDA, HRF, and QM reactions were reported previously [5][6][7][8][9].

Identification of Flavan-3-ols in Strawberry Fruit Sections
For B-type procyanidins composed of (epi)catechin subunits, RDA fragmentation was characterized by the elimination of hydroxyvinylbenzenediol, [M − H − 152] − , and loss an additional molecule of water, [8]. For B-type proanthocyanidins, HRF was characterized by the elimination of 1,3,5-trihydroxybenzene, [M − H− 126] − ion [8]. In some cases, deprotonated 1,3,5-trihydroxybenzene was detected at m/z 125 [8], and this peak was clearly observed in each MS/MS spectrum obtained from the strawberry fruit sections ( Figure 3C-G). The MS/MS spectra of precursor ion at m/z 865.2 are shown in Figure 3F. Figure 4 shows the typical fragmentation pattern of B-type procyanidin, an (epi)catechin trimer. The fragment ion at m/z 713 reflects RDA reaction of an (epi)catechin unit (−152 Da), and the fragment ion at m/z 695 corresponding to the loss of a water molecule (−18 Da). The fragment ion at m/z 739 was caused by HRF reaction of the upper (epi)catechin unit (−126 Da). The fragment ion at m/z 577 corresponds to the (epi)catechin dimer and is known to originate from QM reaction. Again, the fragment ion of the dimer was observed following the RDA-based reaction (m/z 425), which yielded ions at m/z 407 owing to the subsequent loss of a water molecule (−18 Da). The HRF reaction was also observed for the dimer, generating ions at m/z 451. Finally, a QM reaction with the dimer formed a single quinone, resulting in two fragment ions at m/z 289 and 287. In addition, the fragmentation pattern of m/z 865 in strawberry sections was similar to that of the procyanidin C1 standard ( Figure 3F, Supplementary Figure S2G). Therefore, we assigned the peak at m/z 865.2 as B-type (epi)catechin trimer [M − H] − , and the sequence as (epi)catechin-(epi)catechin-(epi)catechin (Table 1). MS/MS analysis of the other potential peaks of B-type procyanidins at m/z 577.1 and 1153.1 clearly showed fragment ions with m/z values matching the expected ions following RDA, HRF, and QM reactions ( Figure 3D,G, Table 1). In addition, the fragment ions of m/z 577.1, and 1153.3 observed in strawberry sections were similar to those of the procyanidin B1 and B2, and cinnamtannin A2 standards, respectively ( Figure 3D Table 1). It is possible that (epi)afzelechin was present in each unit of B-type propelargonidin dimer and trimer. In the MS/MS spectrum of m/z 561.1, we observed neutral losses of 136 (m/z 425) and 152 Da (m/z 409), generated from the precursor ion by RDA reaction (Figures 3C and 4, Table 1). Elimination of ring B from the flavan-3-ol moiety through an RDA reaction with ring C led to the formation of this ion in previous studies [5][6][7][8][9] (Figure 4). The RDA reaction can occur with either the top or base unit. However, elimination of ring B from the top unit gives rise to a fragment ion with a larger π-π-hyper-conjugated system than RDA-based elimination from the base unit of the dimer [5,6]. Hence, it is more energetically favorable. A loss of 136 Da indicated that ring B of the top unit has a single hydroxyl group, whereas a loss of 152 Da indicated that ring B of the top unit has two hydroxyl groups ( Figures 1A and 4). A neutral loss of 126 Da (m/z 435) by HFR reaction was also observed from the precursor ion ( Figure 3C, Table 1). Therefore, we determined the peak at m/z 561.1 to be a B-type propelargonidin dimer [M − H] − ion with one (epi)afzelechin unit, and the sequences as both (epi)afzelechin-(epi)catechin and (epi)catechin-(epi)afzelechin (Table 1). In the MS/MS spectra of m/z 849.2 in strawberry fruit sections ( Figure 3E), the fragment ions at m/z 577 and 561 by QM reaction were observed, suggesting that the top unit of B-type propelargonidin trimer was both (epi)afzelechin and (epi)catechin. In addition, we observed neutral losses of 136 Da (m/z 425) for m/z 561 and 152 Da (m/z 409) for m/z 577 by RDA reaction, suggesting that the middle unit was also both (epi)afzelechin and (epi)catechin ( Figures 3E and 4, Table 1). These results suggested that the base unit was also both (epi)afzelechin and (epi)catechin. A neutral loss of 126 Da (m/z 435 for m/z 561, and m/z 451 for m/z 577) by HFR reaction was also observed ( Figures 3E and 4, Table 1). Therefore, the peak at m/z 849.2 was assigned as B-type propelargonidin trimer [M − H] − ions with one (epi)catechin unit, and the assigned sequences were (epi)afzelechin-(epi)catechin-(epi)catechin, (epi)catechin-(epi)afzelechin-(epi)catechin, and (epi)catechin-(epi)catechin-(epi)afzelechin (Table 1).

Distribution of Identified Flavan-3-ols in Strawberry Fruit
Next, we investigated the distribution of identified flavan-3-ols in strawberry fruit section. As shown in Figure 2A,D-I, (epi)catechin, and B-type procyanidins and propelargonidins, were mainly distributed in the calyx, in and around the vascular bundles and in the skin. Interestingly, the distribution patterns of identified proanthocyanidins between in and around the vascular bundles and in the skin appeared to be different ( Figure 2D-I). To further analyze, we compared the signal intensities of each identified proanthocyanidin between skin and vascular bundles ( Figure 5A). The mean relative intensities of the identified B-type procyanidins in the skin were significantly lower than those in and around the vascular bundles, whereas those of identified B-type propelargonidins showed no significant differences between the skin and vascular bundles ( Figure 5A). Catechin and/or epicatechin showed similar distribution patterns, compared with those of B-type procyanidins ( Figure 5A,B). These results suggested that catechin and/or epicatechin and B-type procyanidins biosynthesis were lower in the skin than in and around the vascular bundles, whereas B-type propelargonidin biosynthesis were almost equal between the tissues [1,2]. Asterisks (*) indicate significant differences between the mean values in the skin and in and around the vascular bundles, as determined by Mann-Whitney U test at the 5% significance level.

Investigation of In-Source Fragmentation of Flavan-3-ols
In our previous study using MALDI-MSI analysis of strawberry fruit sections, in-source fragmentations of anthocyanins were suggested by the detection of anthocyanidins [24]. Therefore, to investigate whether in-source fragmentation of proanthocyanidins occurred, we performed MALDI-MS analyses of the procyanidin standards. In the mass spectra of proanthocyanidin dimers, procyanidin B1, and B2  Figure 6D). These results indicated that in-source fragmentation occurred for all proanthocyanidin standards, by some reactions, i.e., QM reactions in the MALDI-MS analysis. These results also suggested that in-source fragmentation occurred for proanthocyanidins in strawberry fruit sections by MALDI-MSI analysis.

Discussion
In this study, we selected the DAN matrix for analysis after screening various matrices using flavan-3-ol standards and the negative ion mode of MALDI-MSI. We identified (epi)catechin and five proanthocyanidins, namely B-type procyanidin dimer, trimer, and tetramer; and a B-type propelargonidin dimer and trimer with one (epi)afzelechin, using MALDI-MS/MS analysis of ripe strawberry fruit sections. We visualized the identified flavan-3-ols by performing MALDI-MSI analyses, which revealed different distribution patterns between (epi)catechin with B-type procyanidins and B-type propelargonidins in the whole ripe strawberry fruit.
We showed that peaks matching to (epi)catechin monomer or B-type procyanidins were generated from the B-type procyanidins with a larger degree of polymerization by in-source fragmentation ( Figure 6A-D). These results suggested that ion images of identified B-type procyanidins, and B-type propelargonidins in the strawberry fruit sections were mixed ion images of itself and fragment ions generated from the B-type procyanidins, and B-type propelargonidins with a larger degree of polymerization ( Figure 2D-I). Previous studies [31,32] using LC-MS/MS reported that (epi)afzelechin was located on the top unit of the B-type propelargonidin dimer and trimer with one (epi)afzelechin in strawberry fruit ( Figure 5); however, using MALDI-MS/MS, in this study (epi)afzelechin was also located at the base unit of the dimer, and middle or base units of the trimer, respectively (Table 1). This discrepancy might be due to the different strawberry species used here or to the in-source fragmentation of proanthocyanidins. To visualize each flavan-3-ol by itself using MSI, it may be necessary to develop a softer ionization mass-spectrometer than the vacuum MALDI-TOF/TOF used in this study.
Our data showed that the relative amounts of B-type procyanidins were significantly higher in and around the vascular bundles than in the skin, whereas comparable amounts of B-type propelargonidins were found between in and around the vascular bundles and in the skin ( Figure 5A). Although, in the biosynthesis of proanthocyanidins, it has not been fully determined the biosynthesis mechanisms, for example, whether the biosynthesis is enzymatic or not [1], it is known that proanthocyanidins are formed through the condensation of flavan-3-ol units such as (epi)catechin and (epi)afzelechin [1,2]. Procyanidins are biosynthesized from (epi)catechin only, whereas propelargonidins are biosynthesized from both (epi)catechin and (epi)afzelechin [1]. As shown in Figure 5B, the amount of (epi)catechin in and around the vascular bundles was significantly higher than that in the skin. Based on these results, we speculated that the distribution patterns of procyanidins and propelargonidins depended on the distribution patterns of their flavan-3-ol monomers. Therefore, the lack of a significant difference in propelargonidin levels between in the skin and in and around the vascular bundles in strawberry fruit might be caused by smaller differences in the amounts of (epi)afzelechin between in the skin and in and around the vascular bundles, compared with the corresponding (epi)catechin levels.
In angiosperms, flavan-3-ols help protect against ultraviolet radiation and ozone by reducing oxidative stress [1]. Flavan-3-ols can play a key role in the protection against mammals and plant-feeding insects [1]. Tannins have been shown to be toxic to bacteria [1,33]. In addition, flavan-3-ol monomers and proanthocyanidins may also inhibit the germination of fungal spores and block the biosynthesis of melanin, which is an important factor in the virulence of many pathogenic fungi attacking plants [1,34]. Nizioł et al. [35] visualized the only proanthocyanidin, gambiriin C (epiafzelechin-catechin) using MSI with 109 Ag nanoparticle enhanced target, and showed that the proanthocyanidin was mainly distributed in the skin and achenes of strawberry fruit. They reported that the distribution of flavan-3-ols in strawberry fruit was related to their protective function. In the present study, we revealed that flavan-3-ols are mainly distributed not only in the skin but also in the calyx and in and around the vascular bundles. Pathogens such as bacteria and fungi infect plant surface tissues such as the fruit skin, which proliferate in the infected tissues and immediately spread throughout the plant body through fluid in vascular bundles. Therefore, flavan-3-ols, especially (epi)catechin and B-type procyanidins, might also be present in and around the vascular bundles of ripe strawberry fruit to prevent the infection and spread of pathogens.
Although the biological significance of the different distribution patterns between B-type procyanidins and propelargonidins is currently not well understood, distribution analysis of flavan-3-ols in strawberry fruit under different abiotic and biotic stress conditions will improve our understanding of the biological significance of flavan-3-ols in strawberry fruit [1][2][3]. In addition, the spatial information would assist those working in the field of breeding to enhance the prevention effects of flavan-3-ols against chronic diseases by improving the genetic properties of strawberries [24,36]. Furthermore, the MALDI-MSI analysis used in this study might be adapted to investigate the distribution of flavan-3-ols in various plant species and tissues, except strawberry fruit. Recently, pharmacokinetic analyses, i.e., post-consumption tissue localization analyses of polyphenols such as anthocyanins using MALDI-MSI, have been performed [37]. Therefore, the MALDI-MSI analyses used in this study would be also helpful to investigate the localization of PAs in animal tissues for the elucidation of their health-promoting mechanisms. In conclusion, we demonstrated that the MALDI-MSI analysis used in this study is a valuable tool for distribution analysis of flavan-3-ols in strawberry fruit.

Strawberry (Fragaria × Ananassa Duch.) Samples
'Tochiotome' strawberry fruit, a popular cultivar in Japan, were cultivated in the Strawberry Research Center (Tochigi, Japan). Ten ripe strawberries were harvested, frozen on the day of harvest, and stored at −80 • C until use.

Preparation of Fruit Sections
Fruit sections were prepared using CMC freeze-embedding, as described in our previous study [24]. Briefly, the fresh fruit were immersed in CMC (2%) and flash-frozen in liquid nitrogen. Subsequently, longitudinal and cross-sections (100 µm thick) were consecutively prepared using a CM 1860 cryostat (Leica Microsystems, Wetzlar, Germany). The sections were then mounted onto ITO-coated glass slides and placed in 50 mL conical centrifuge tubes containing silica gel for drying. The sections were preserved at −80 • C in a freezer until MALDI-MSI analysis was performed.

MALDI-MSI Analysis
MALDI-MSI analysis was performed according to our previous study [24], with minor modifications. The flavan-3-ol standards and strawberry fruit sections were analyzed using a MALDI-TOF/TOF instrument (UltrafleXtreme, Bruker, Billerica, MA, USA) equipped with a 355 nm Nd:YAG laser, using a repetition rate of 1000 Hz. Data were acquired using a step size of 300 µm in negative-ion mode (reflector mode). The m/z values in the range of 240-1200 were measured. The laser diameter was set to the medium size. The spectra were acquired automatically using FlexImaging 4.1 software (Bruker). Normalization of spectra based on the total ion current was performed using the same software. The FlexImaging 4.1 software was also used to create two-dimensional ion-density maps and for peak analyses.
To analyze the strawberry fruit sections, frozen sections were took out from a freezer and dried in a vacuum desiccator for 30 min. Six milliliters of a DAN solution (10 mg/mL in 80% aqueous methanol) was sprayed uniformly over the section using a 0.18 mm nozzle caliber airbrush (Mr. Airbrush Custom Double Action; Mr. Hobby, Tokyo, Japan), after which MALDI-MSI analysis was performed. To investigate the spatial distribution of the identified proanthocyanidins, three different strawberry fruits were analyzed. The mass spectra and ion images of the identified flavan-3-ols in the three different strawberry fruits showed similar patterns (Supplementary Figure S3). The mass spectrum and ion images of one of the three different strawberry fruits are presented as representative data in Figure 2C-I.
To compare the intensities of the identified flavan-3-ols between the skin and vascular bundles, three sections of the same strawberry fruit were analyzed and their tissue-specific intensities were obtained using the region-of-interest function of FlexImaging 4.1 software. These data are shown in Figure 5.

MALDI-MS/MS Analysis of Strawberry Fruit Sections
MALDI-MS/MS analysis was performed according to our previous study, with minor modifications [24]. Briefly, the MS/MS spectra were obtained from the strawberry sections after performing MALDI-MSI analysis with an ultrafleXtreme instrument operated in collision-induced dissociation "LIFT" MS/MS mode. The m/z values of precursor ions were set at each value ±1%.
The MS/MS spectra were analyzed using FlexAnalysis 3.4 software (Bruker). The molecular species of proanthocyanidins detected from the sections were assigned based on typical neutral losses observed after retro-Diels-Alder (RDA), heterocyclic ring fission (HRF), and quinone methide (QM) reactions [7][8][9], on comparison with the MS/MS spectra of the proanthocyanidin standards.