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Imaging mass spectrometry analysis of flavonoids in blue viola petals and their enclosure effects on violanin during color expression Kohtaro Sugahara, Kazunori Kitao, Takehiro Watanabe, and Tohru Yamagaki Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03815 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Analytical Chemistry

Imaging mass spectrometry analysis of flavonoids in blue viola petals and their enclosure effects on violanin during color expression Kohtaro Sugahara,* Kazunori Kitao, Takehiro Watanabe, and Tohru Yamagaki* Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1 Seikadai, Seika-cho, Soraku-gun, Kyoto 6190284, Japan ABSTRACT: The color expression of anthocyanin pigments in blue flowers is precisely controlled by their chemical and physical properties such as pH and the presence of metal ions or colorless copigments. Despite the large number of known blue flowers, their coloration mechanisms have not been examined in sufficient detail. In this work, the blue coloration of Viola cornuta petals was expressed via the copigmentation of various flavonol 3-O-glycosides. By using a combination of imaging mass spectrometry with liquid chromatography mass spectrometry, the structures and contents of flavonols co-localized with violanin in the discrete bluecolored regions of the petal were identified. The obtained data allowed the in vitro reconstruction of the color expression that was consistent with the visible spectrum of the viola petal. The results of visible spectral analysis indicated that neither the increase in the solution pH inside the vacuole cells nor the presence of metal ions affected the color development process. Ultimately, it was experimentally confirmed that the excess amounts of flavonol 3-O-glycosides complexed with violanin, which prevented violanin molecules from forming a levorotatory helical self-assembly during the blue color expression via copigmentation.

Colors and patterns of flower petals originate from the presence of anthocyanins in their vacuole cells. In particular, delphinidins, pelargonidins, and cyanidins are found in blue, orange, and red petals, respectively.1–6 However, depending on the specific chemical and physical parameters of the vacuoles, the same anthocyanins can express different colors ranging from red to blue.1,2 Generally, anthocyanins that can be isolated in the form of red flavylium salts using acidic solvents are unstable under neutral or weakly acidic conditions because of their immediate hydration leading to the formation of colorless chalcones (Figure S-1).3 Nevertheless, the color of anthocyanins in flower petals remains stable although the pH of the solution inside their vacuoles usually ranges from 5 to 6.5. Since Willstätter and Everest investigated the effects of the vacuole solution pH,4 the chemical stability and color variation of anthocyanins in flowers have attracted considerable attention.5 While a number of anthocyanin pigments are directly responsible for the blue color expression in flower petals, various factors including their structures, self-stacking process, complexation with metal ions, presence of colorless substances called copigments, and structural changes caused by the variations of the solution pH inside vacuoles significantly affect the tone of blue coloration. Nevertheless, the copigmentation phenomenon remains poorly understood.3,6 More recently, imaging mass spectrometry7–9 (MS) has been utilized to detect various plant tissue metabolites such as carbohydrates,10 organic acids,11 lipids,12 proteins,13 terpenes,14 alkaloids,15 glucosinolates,16,17 and polyphenols.18 In addition, this method was also employed for the quantitative evaluation of metabolite contents in the regions of interests (ROIs). However, despite its versatility, this technique has some disadvantages caused by various factors such as the heterogeneity of the tissue surface, ion suppression by coexisting substances, and sample preparation method on the

quality of imaging MS spectra.19–23 To resolve these problems and develop a reliable quantitative evaluation method for imaging MS, two different techniques, namely matrix-assisted laser desorption imaging (MALDI) MS and liquid chromatography-coupled electrospray ionization (LC-ESI) MS quantification were combined in this work. The content of each metabolite quantified using the developed method was distributed across all pixels of the tissue surface according to the normalized ion counts.24–26 To elucidate the molecular mechanism of the blue flower coloration via copigmentation, the blue petals of Viola cornuta, which contained one anthocyanin pigment (violanin) and a number of flavonols acting as potential copigments were examined. To determine the structures and contents of copigments in the blue part of V. cornuta petals, multiple compounds must be identified and quantified simultaneously. Therefore, in this work, a matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) instrument was used for the analysis of the adaxial petal surface via imaging MS as pioneered by Caprioli.7–9

Experimental Section Plant material. Petals of openly cultured V. cornuta (Fiolina Skyblue, Suntory Flowers Limited, Japan) harvested in seven days after anthesis were used in imaging MS, LC-MS quantification, and microscopic spectral experiments. Imaging MS studies. A cultivated viola petal was directly placed between two glass slides and dried in vacuo. The dried petal was then pasted on an indium tin oxide coated glass slide (Bruker Daltonics, Germany) using conductive adhesive tape (Shimadzu, Japan) for the subsequent analysis of its adaxial face with an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany). A methanol/water solution of 2,5-dihydroxybenzoic acid (DHB, Sigma-Aldrich, USA) was

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sprayed uniformly over the petal surface using an airbrush with a 0.2-mm nozzle (Procon Boy FWA Platinum, Mr. Hobby, Japan). The laser irradiation focus, pitch, and number of shots per pixel were set to 5 (ultra large), 150 µm, and 300, respectively, using the flexImaging software (Bruker Daltonics, Germany). All imaging MS spectra recorded in this study were processed by conducting a total ion count normalization procedure and then converted to a commonly used multidimensional imaging MS data format (imzML) for constructing ion-content maps.27 The MsIQuant software developed by Källback and co-workers28 was utilized to extract the spatial information (X, Y) and MS signal intensities (Ii in Eq. (1)) for individual flavonoids in the studied regions. In order to create ROIs for a color in the petal sample, the RGB color model was employed. RGB values of each ROI were extracted from the scanned optical images of the flowers using Microsoft PowerPoint 2016® software. Each ROI was constructed in accordance with the RGB values listed in Table S-2. LC-MS quantification of flavonoids. Both single and sectioned viola petals were weighed, homogenized with zirconia beads (i.d.: 5.0 mm) in a 2.0-mL plastic tube using a bead beater (Tissue Lyser II, QIAGEN GmbH, Germany), and sequentially extracted with 2 mL of 50 vol.% aqueous acetonitrile solution containing 0.1 vol.% trifluoroacetic acid and 2 mL of methanol. Before LC-MS analysis, the obtained extract was passed through a membrane filter with a pore size of 0.45 μm (Cosmo nice filter, Nacalai Tesque, Japan). A 10μL sample of the obtained extract was subjected to LC-MS quantification. An LCMS-8030 triple-quadrupole mass spectrometer (Shimadzu Corporation, Japan) was used for the LC-MS quantification of violanin and flavonols in the viola petals. The mass spectrometer was coupled with a Nexera highperformance liquid chromatograph (Shimadzu Corporation, Japan), and all data analyses were performed using the LabSolutions software (version 5.53, Shimadzu Corporation, Japan). Samples were separated on a reverse phased column (Cosmosil 5C18 AR-II, 2.0 mm i.d. × 150 mm, Nacalai Tesque, Japan) with the following solvent gradient: 10–25 vol.% B for 0–15 min, 25–80 vol.% B for 15–20 min, and 10 vol.% B for 20–27 min, where solvent A was a 0.1% aqueous solution of formic acid, and solvent B was a 0.1% formic acid solution in acetonitrile (A + B = 100 vol.%). The flow rate was set to 0.15 mL/min, and the mass spectrometer was operated in the selective ion monitoring mode for both negative and positive ions. Flavonoid quantification was performed by measuring the areas of the recorded LC-MS peaks and comparing them with the corresponding points on the external calibration curves obtained for each flavonoid. In vivo visible spectra acquisition. Microscopic visible spectra were recorded in the transmission mode on a MSV-5200 spectrophotometer (JASCO Corporation, Japan) in the wavelength range of 400–800 nm. The diameters of the “In” and “Out” apertures were set to 100 and 200 μm, respectively. A peeled layer of viola petal containing epithelium cells was placed between two KBr plates, and the resulting void was filled with glycerol to reduce the light scattering. After that, the petal was loosely pressed to preserve its vacuoles. In vitro visible spectra acquisition. Visible spectra were recorded on a V-650 UV-visible spectrophotometer (JASCO Corporation, Japan) in the wavelength range of 400–800 nm at ambient temperature using a quartz cell with a light path length

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of 0.1 mm. The concentration, solvent type, and molar absorption intensities of each flavonoid are listed in Table S-2. Reconstruction experiments. Circular dichroism (CD) spectra were recorded in the wavelength range of 400–800 nm on a J-725 spectrodichrometer (JASCO Corporation, Japan) at ambient temperature using a quartz demountable cell with a 0.1mm light path. The pH of the media was set to a value of 5.8, which was determined with a pH meter equipped with a microelectrode (N = 3, Ave., F-22 with a 9618S-10D electrode, Horiba, Ltd., Japan) during petal squeezing. This procedure was performed under N2 atmosphere to exclude possible interferences of atmospheric CO2 species. After examining the effects of Fe3+ ions on the blue color expression, it was found that their addition into the phosphate buffer resulted in precipitation. Hence, violanin and flavonols were dissolved in 0.1 M McIlvaine buffer solution containing 5.3 mM of FeNH4(SO4)2 to avoid precipitate formation. All other substrates were dissolved in 0.1 M potassium phosphate buffer.

Results and Discussion Dominant flavonoids accumulated in the blue viola petal. The absolute structures of the dominant flavonoids present in the petal of V. cornuta are shown in Figure 1 (see also Figures S-3–S-15 and Tables S-1–S-2). The petal consisted of violanin, which represented the only anthocyanin pigment, a number of flavonol 3-O-glycosides, and apigenin 6,8-di-C-glycoside (called violanthin). Moreover, M3GRRMe and Q3GRRmalo species were isolated as novel flavonol 3-O-glycosides. Construction of pixel maps for each flavonoid in the viola petal by imaging MS. To determine whether flavonols could act as copigments in the blue color expression of the petal, its structural information (including the spatial distributions of flavonoids) was obtained by the imaging MS analysis of the intact petals. However, a direct comparison of the distributions of different flavonoids by imaging MS requires a valid method for converting the MS signal intensity of each metabolite into its absolute content in pixels (pmol/pixel). For this purpose, we developed an alternative quantitative calculation technique based on the strategy proposed by Hattori and co-workers24 since it was found suitable for the simultaneous analysis of multiple compounds without a standard.22, 23 The content of each flavonoid (CLC-MS) in a half of the petal sheet was proportional to the MS pixel intensity according to the following formula:

Ci=CLC-MS·Ii/It

(1)

Here, Ci represents the flavonoid content in the ith pixel [pmol/pixel], CLC-MS is the total content in the half petal determined by LC-MS [pmol], Ii is the MS signal intensity per pixel [arbitrary units], and It is the total MS signal intensity obtained for the petal half [arbitrary units]. The resulting MS images (Figures 2A, B, and C) contain the spatial distributions of individual flavonoids on heat maps. From the obtained pixel maps (Figures 2A, B, and C), the flavonoid contents in the four ROIs were determined according to Eq. (2), which represented a modified version of Eq. (1) (see Figures 3 and 4).


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Analytical Chemistry

CROI=CLC-MS·IROI/It

(2)

Here, CROI is the content of each flavonoid in the respective ROI [nmol], CLC-MS is the total content in the half petal obtained by LC-MS [nmol], IROI is the MS intensity in the corresponding ROI of the petal [arbitrary units], and It is the MS intensity obtained for the half petal [arbitrary units]. As shown in Figure 4, the flavonoid contents obtained by imaging MS was in good agreement (within an order of magnitude) with the values determined by the LC-MS analysis of the sectioned petal tissues (see Figure 3).

Figure 1. Dominant flavonoids accumulated in the blue viola petal. The utilized abbreviations are Q: quercetin, M: myricetin, K: kaempferol, T: tamarixetin, iR: isorhamnetin, G: O-β-Dglucopyranose, R: O-α-L-rhamnopyranose, Me: methyl, malo: malonate, 3: 3-O-glycoside, and 7: 7-O-glycoside.

Heterogeneous distribution of flavonoids over the petal surface. As shown in Figure 2D, four ROIs were defined using different colors [deep blue (DB), light blue (LB), nectar guide (NG), and yellow (YL)] to determine which particular flavonoids were present in the blue petal parts. To distinguish each ROI according to its color, the MS images presented above illustrate the dependence of the formation of heterogeneous distribution patterns on the types of aglycone or sugar substituents (Figure 2). In particular, violanin was consistently localized in the bluish DB and NG regions (Figures 2C, S-16, and S-18). Consistent results of negative ion measurements were obtained for most flavonoids (Figures 2A and S-16). Negative ion MS/MS measurements targeting the precursor ion

with m/z = 771 produced distinct ion images for m/z = 316 ([myricetin–H•–H]–) and m/z = 301 ([quercetin–H]–), corresponding to M3GRR and Q3GR7G species, respectively (Figures 2B and S-17). In contrast, positive ion measurements resulted in clear images for both violanin and M3GRRMe compounds because this mode produced stronger signals as compared to those obtained during negative ion measurements (Figures 2C and S-18). As a result, a complete set of petal MS images was successfully acquired with sufficient reproducibility (N = 5, Figure S-19). The obtained data indicate that Q3G, M3GR, and M3GRR compounds acted as copigments as they were strongly colocalized with violanin in the NG or DB regions. Determination of flavonoid concentrations from imaging MS data. From the obtained contents of individual flavonoids and water in petal tissues, the flavonoid molar concentrations in the NG section were calculated in the following decreasing order: Q3GR (26.79 mM), Q3GRR (11.61 mM), M3GRR (2.43 mM), M3GR (2.06 mM), Q3GR7G (0.54 mM), M3GRRMe (0.49 mM), T3GRR (0.23 mM), K3GR (0.14 mM), Q3GRRmalo (0.12 mM), K3GRR (0.11 mM), iR3GR (0.08 mM), and Q3G (0.01 mM). Among these flavonols, Q3GR, Q3GRR, M3GRR, and M3GR were examined in more detail due to their abundancy and, therefore, relatively strong effect on the color expression. In vitro reconstruction of the blue coloration of the viola petal. To verify the participation of flavonols in the blue color expression as copigments, the in vivo state of pigment cells was investigated first by examining the microscopic visible spectra of the NG section in the detached petal (Figures 5A, C, and D).31 Afterwards, the blue color of this section was reconstructed by mixing the dominant flavonols (with contents above 1 mM) and violanin at ratios calculated in the previous section (Figure 5B). In both the in vivo and reconstructed spectral analyses, the pH value was equal to 5.8, which corresponded to the pH of the vacuoles of cultivated petals obtained in 7 d after anthesis (see the Experimental Section). The reconstructed state contains almost 10 equivalents of multiple flavonols with respect to violanin (Figure 5B). In order to verify whether multiple flavonols were required for the color expression, we created a simplified experimental system by replacing all flavonols with M3GRR, which was referred to as “simplified system” in Figures 5 and 6. The maximum absorption wavelength and spectral shape of the peeled petal matched very well the parameters obtained for both the reconstructed states and simplified system at wavelengths of around 550, 580, and 630 nm and pH = 5.8 (Figure 5A). These results explicitly indicated that the blue color development caused by the presence of flavonol 3-O-glycosides did not depend on their aglycone or sugar moieties and that the skeletal structure of the flavonols was responsible for the blue color expression of violanin through copigmentation.



Figure 2. MS images of the dominant flavonoids in the petal. (A) Negative ion images. (B) Negative ion MS/MS images. (C) Positive ion MS images. The corresponding optical images in panels (A), (B), and (C) are displayed on the right sides. Individual flavonoid contents in a single pixel [Ci in Eq. (1)] are expressed on the heat maps in [pmol/pixel]. (D) Optical image of the viola petal obtained via negative ion measurements, in which four different ROIs (DB, LB, NG, and YL) were created. The lengths of all scale bars are 5 mm.

Figure 3. Photographs describing the sectioning of a viola petal along the white dashed line into four types of tissues with different colors: DB, LB, NG, and YL. Bar length = 5 mm.

Figure 4. Comparison of the dominant flavonoid contents in the sectioned petal obtained by the imaging MS (empty columns) and LC-MS (filled columns) techniques. The data are presented as mean ± SD, N = 5.


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Analytical Chemistry coloration of the copigmentation.31, 32

Figure 5. (A) Visible spectra of the nectar guide obtained through the reconstruction of the imaging MS data (solid line) and actual microscopic spectrum (dotted line). (B) Concentrations of the dominant flavonols (above 1 mM) obtained by imaging MS. All flavonoids were dissolved in 0.1 M phosphate buffer with pH = 5.8, which was equal to the pH of the petal squeeze. (C) Optical image of the viola petal also depicted in Fig. 3, in which the NG region is denoted by the white arrow. (D) Microscopic image of a viola petal, in which the studied regions are surrounded by the white circles.

Effects of pH and presence of metal ions on the blue coloration of violanin. Apart from copigmentation, the presence of Fe3+ ions and pH of the vacuolar media equal to 8.0 were previously reported as significant factors affecting the blue color expression.6 Thus, the spectroscopic features of the reconstructed state were compared with those obtained at high pH or in a medium containing Fe3+ species. Relatively large wavelength shifts (known as bathochromic shifts) were observed in the reconstructed state as compared to those of pure violanin at pH = 5.8 (Figure 6A). The fact that violanin turned blue and exhibited two peaks (centered at 555 and 661 nm) at pH = 8.0 and three peaks (centered at 548, 576, and 629 nm) at pH = 5.8 suggested that its quinonoidal form was dominant in the solution with a pH of 8.0. Notably, violanin also became blue at pH = 5.8 with the addition of copigments; however, the corresponding absorption wavelengths significantly differed from those obtained at pH = 8.0, presumably due to the positions of the quinone moieties in violanin molecules (see Figure S-1). It is interesting that the addition of Fe3+ ions caused the appearance of broad peaks in the range of 580–750 nm regardless of the presence of copigments, owing to the absorption induced by the ligand-to-metal charge transfer between Fe3+ and succinylcyanin species.30 Furthermore, elemental distributions on the petal surface were analyzed using an electron probe microanalyzer (EPMA, Figure S-20). The obtained distributions did not match the shape of the nectar guide in the petal. These results confirmed that the metal ions were not related to the blue color expression and that the blue

viola

petal

was

developed

via

Figure 6. (A) Visible and (B) CD spectral changes of violanin induced by copigmentation, high pH, and the presence of metal ions. (C) Measured flavonoid concentrations. When Fe3+ ions were added to flavonoids, the corresponding samples were dissolved in 0.1 M McIlvain buffer. For all other specimens, 0.1 M phosphate buffer solution was used.

Conformational changes of violanin during copigmentation under physiological conditions. Violanin exhibited a strongly negative first Cotton effect at both pH = 5.8 and 8.0 (Figure 6B), which indicated the formation of a counterclockwise helical self-stacking structure under these conditions, as was previously reported for other anthocyanins.33 These results suggest that violanin did not form a self-stacking structure in the presence of copigments under physiological conditions since the negative Cotton effect was not observed for the reconstructed state. The visible spectral pattern of the simplified system (Figure 5A) suggests possible reconstruction of the blue coloration when the content of copigments (such as flavonol 3-Oglycosides) was maintained at a level of 10 equivalent fractions of violanin (Figure 5B). Therefore, the concentration dependence of M3GRR on the structure of violanin in solution was examined. Figure 7A illustrates the CD spectra recorded after the addition of 0.5–50 equivalents of M3GRR to violanin at pH = 5.8. It shows that the amplitude of the first Cotton effect decreased with increasing M3GRR content and becomes zero when the latter reaches 50 equivalents.



Figure 7. (A) Effects of flavonol M3GRR on the self-stacking of violanin evaluated from the observed decrease in the amplitude of the Cotton effect with the addition of M3GRR (0–50 eq.) to the violanin solution. (B) Inhibitory effect of the copigment on the violanin degradation observed from the decay of violanin absorbance with time before and after the addition of ten equivalents of M3GRR. Here, I0 and I denote the absorbance values measured at the initial and given points, respectively. (C) A schematic describing possible interactions between violanin and flavonol species: the mechanisms of the molecular association and color change reactions of the violanin solutions with and without flavonols. functional analyses of flavonoids in plants (including color expression via copigmentation). Protective effects of flavonol 3-O-glycosides on violanin under physiological conditions. Figure 7B shows the time ASSOCIATED CONTENT decay plots of the maximum absorbance ratios of the violanin solutions with and without M3GRR (measured at λmax = 556 and Supporting Information 526 nm, respectively). A significant fraction (32%) of violanin Detailed materials and methods, possible structures of was spontaneously decomposed into colorless compounds after anthocyanins in solution (Figure S-1), RGB color values for 120 min of reaction, whereas the addition of 10 equivalents of creating ROIs (Figure S-2), physicochemical properties of the M3GRR reduced this magnitude to less than 5%. From these flavonoids used in this study (Tables S-1 and S-2, Figures S-3–Sresults, it can be concluded that the presence of M3GRR species 15), averaged imaging MS spectra and signal assignment of 1) shifts the visible absorption spectrum of violanin to longer negative ions, negative MS/MS images, positive ion measurements wavelengths; 2) inhibits the self-stacking of violanin molecules; (Figures S-16–S-18), five individual MS images of Q3GRR, and 3) protects violanin chromophores from hydration (see violanin and M3GRR (Figure S-19), and 2D-EPMA images of the Figure 7C). petal (Figure S-20). The Supporting Information is available free of charge on the ACS Publications website.

Conclusion The present study was conducted to elucidate the blue color expression mechanism of blue viola petals. While investigating the properties of anthocyanin and co-pigments, a quantitative interpretation method for imaging MS was developed, which provided both the structures and contents of violanin and flavonol 3-O-glycosides in the NG section of blue petals. From the good agreement between the in vivo and reconstructed state spectra, it was found that the blue coloration of viola petals was expressed via the copigmentation of flavonol 3-O-glycosides and violanin. In addition, a simplified reconstructed system was obtained by replacing multiple flavonol 3-O-glycoside species with M3GRR, which enabled the assessment of both the protective effect of copigments and specific CD spectral changes during copigmentation. Hence, it was concluded that the self-stacking of violanin did not occur inside the vacuole; instead, the excessive flavonol 3-O-glycoside molecules surrounded the violanin chromophore, thereby loosening its helical structure. The analytical methods developed in this work and obtained data can be used in future studies involving

AUTHOR INFORMATION Corresponding Authors *E-mails: [email protected], [email protected]

Author Contributions K. S. and T. Y designed the project; K.S. and K. K. performed the experiments; K. S., K. K., and T. W. analyzed the data; K. S. and T. Y. wrote the manuscript. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank Mr. Naoto Kambayashi from JASCO Engineering for conducting the microscopic analysis as well as Mr. Kazunori Kitajima and Mr. Satoshi Yoshimi from SHIMADZU Corporation for performing the EPMA measurements. We are also grateful to Dr. Hiroyuki Minakata and Shigetada Nakanishi (Suntory


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Analytical Chemistry Foundation for Life Sciences) as well as to Prof. Makoto Suematsu (Keio University) for fruitful discussions.

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