Different Localization Patterns of Anthocyanin Species in the Pericarp of Black Rice Revealed by Imaging Mass Spectrometry

Kawamura Y (2012) Different Localization Patterns of Anthocyanin Species in the Pericarp of Black Rice Revealed by Imaging Mass Spectrometry. PLoS ONE 7(2): e31285. doi:10.1371/journal.pone.0031285 Different Localization Patterns of Anthocyanin Species in the Pericarp of Black Rice Revealed by Imaging Mass Spectrometry Yukihiro Yoshimura 0 Nobuhiro Zaima 0 Tatsuya Moriyama 0 Yukio Kawamura 0 Gustavo Bonaventure, Max Planck Institute for Chemical Ecology, Germany 0 Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University , Nara , Japan Black rice (Oryza sativa L. Japonica) contains high levels of anthocyanins in the pericarp and is considered an effective health-promoting food. Several studies have identified the molecular species of anthocyanins in black rice, but information about the localization of each anthocyanin species is limited because methodologies for investigating the localization such as determining specific antibodies to anthocyanin, have not yet been developed Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a suitable tool for investigating the localization of metabolites. In this study, we identified 7 species of anthocyanin monoglycosides and 2 species of anthocyanin diglycosides in crude extracts from black rice by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. We also analyzed black rice sections by MALDI-IMS and found 2 additional species of anthocyanin pentosides and revealed different localization patterns of anthocyanin species composed of different sugar moieties. Anthocyanin species composed of a pentose moiety (cyanidin-3-O-pentoside and petunidin-3-O-pentoside) were localized in the entire pericarp, whereas anthocyanin species composed of a hexose moiety (cyanidin-3-O-hexoside and peonidin-3-O-hexoside) were focally localized in the dorsal pericarp. These results indicate that anthocyanin species composed of different sugar moieties exhibit different localization patterns in the pericarp of black rice. This is the first detailed investigation into the localization of molecular species of anthocyanins by MALDI-IMS. - Rice (Oryza sativa L.) is one of the most important cereal crops in the world, especially in Asian countries. One of the various rice cultivars is black rice, which contains high levels of anthocyanins in the pericarp and is considered an effective health-promoting food. The crude anthocyanin-rich extracts of black rice bran improve serum triglyceride levels, which contributes to the suppression of atherosclerosis [1,2], and protects against insulin resistance [3], alcoholic liver injury [4], and light-induced retinal damage [5,6]. Due to these human health benefits, anthocyanin compositions have been investigated not only in black rice but also in various plants, such as berries [7], black soybeans [8,9,10], and darkcolored grapes [11,12]. Several studies of the molecular species of anthocyanins in black rice have identified cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, peonidin-3-O-glucoside, and cyanidin-3O-rutinoside [13,14,15,16]. However, there are virtually no localization studies of individual anthocyanin species in plant tissue because methodologies for investigating the localization, such as determining specific antibodies to anthocyanin, have not yet been developed. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging technology that allows the simultaneous investigation of the content and spatial distribution of a wide range of biomolecules such as lipids [17,18,19], glycolipids [20,21], amino acids [22], proteins [23], and peptides [24,25], as well as administered pharmaceuticals [26] without requiring antibodies, staining, or complicated pretreatment steps. Our previous study demonstrated that MALDI-IMS combined with the Kawamoto method, which uses adhesive films to prepare the cryosections, is an effective method for investigating the localization and composition of rice metabolites [27]. In this study, we investigated the localization of several anthocyanin species in black rice tissue in detail by MALDI-IMS and revealed the different localization patterns of anthocyanin species in the pericarp of black rice. Identification of anthocyanins in the crude extract from black rice seed Figure 1A is an image of a whole black rice seed used in this study. A 10-mm- thick cryosection shows that the black or purple pigment is localized in the pericarp and seed coat layers (Fig. 1B, C) and that the pericarp and aleurone layers were thicker in the dorsal side than in the ventral side (Fig. 1B). We used MALDI-MS to analyze the crude extract to identify the anthocyanins in the pigmented layers of black rice. As shown in Figure 1D, abundant anthocyanin signals at m/z 419, 433, 449, 463, 465, and 479 were observed in the mass spectrum. We analyzed precursor ions by tandem mass spectrometry (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 demonstrated an intense ion at m/z 287 (neutral loss of 132 Da), which would correspond to the loss of a pentose moiety (Fig. 2A). Unfortunately, the MALDI-MS system could not differentiate between molecular species of sugar moieties that have the same molecular mass, such as xylose and arabinose. The MS/MS fragmentation of the ion at m/z 287 would correspond to the cyanidin aglycone. Thus, we identified the m/z 419 ion as an [M]+ ion of cyanidin-3O-pentoside (Fig. 2A). The MS/MS spectrum of the m/z 433 ion demonstrated an ion at m/z 271 (neutral loss of 162 Da), which would correspond to the loss of a hexose moiety (glucose or galactose), and the product ion at m/z 271 ion would correspond to the pelargonidin aglycone (Fig. 2B). Therefore, we identified the m/z 433 ion as an [M]+ ion of pelargonidin-3-O-hexoside (Fig. 2B). Similarly, because the ions in the black rice crude extract at m/z 449, 463, 465, and 479 also yielded the characteristic neutral loss of 162 Da corresponding to the loss of a hexose moiety, these ions were identified as the [M]+ ions of cyanidin-3-O-hexoside (Fig. 2C), peonidin-3-O-hexoside (Fig. 2D), delphinidin-3-Ohexoside, and petunidin-3-O-hexoside (Table 1), respectively. The molecular species identified in MS/MS analyses are summarized in Table 1. Recently, cyanidin-3-O-gentiobioside consisting of 2 glucose moieties and a cyanidin aglycone was identified in black rice [28]. We performed MS/MS analyses on the m/z 611 and 625 precursor ions to examine the possibility that this anthocyanin diglycoside might be included in the black rice crude extract used in this study. MS/MS analysis of the precursor ions at m/z 611 yielded an ion at m/z 287 corresponding to the aglycone cyanidin, which could be formed by a neutral loss of 2 hexoses (Fig. 2E). A small peak detected at m/z 449 indicated the possibility that the ions for (...truncated)