Figure 1.
Identification of anthocyanin species in black rice crude extract.
A, Appearance of black rice. B, Black rice section stained with toluidine blue O. Scale bar: 1.0 mm. C, Enlarged image of the dorsal region of the longitudinal black rice section. D, Mass spectrum of the black rice crude extract.
Figure 2.
Representative MS/MS spectra of ions in black rice crude extract.
A–D, [M]+ ions at m/z 419 (A), m/z 433 (B), m/z 449 (C), and m/z 463 (D). Neutral losses (NL) of 132 Da and 162 Da indicate losses of a pentose and hexose, respectively. E, F, [M]+ ions at m/z 611 (E) and MS3 spectrum of ions at m/z 449 derived from ions at m/z 611 (F).
Table 1.
Identified anthocyanin species in this study.
Figure 3.
MALDI-IMS analysis of anthocyanins in black rice sections.
A, Black rice section stained with toluidine blue O after MALDI-IMS analysis of the ions at m/z 449. B, Localization pattern of the ions at m/z 449. C, Localization pattern of the ions at m/z 822 corresponding to PC (diacyl C36:3), which marks the nucellus epidermis/aleurone layer. D, Localization pattern of the ions at m/z 496 corresponding to LPC (C16:0), which marks the endosperm. E, Merged ion image of m/z 449 (red), m/z 822 (green) and m/z 496 (white). F, Black rice section stained with toluidine blue O after MALDI-IMS analysis of the ions at m/z 463. G, Localization pattern of the ions at m/z 463. H, Localization pattern of the ions at m/z 822 corresponding to PC (diacyl C36:3). I, Localization pattern of the ions at m/z 496 corresponding to LPC (C16:0). J, Merged ion image of m/z 463 (red), m/z 822 (green) and m/z 496 (white). Scale bar: 1.0 mm.
Figure 4.
MALDI-IMS/MS analysis of the ions at m/z 449 and m/z 463.
A, B, MS/MS spectrum of ions at m/z 449 (A) and m/z 463 (B) obtained from the pericarp of a black rice section. C, Optical image of a black rice section after analysis of the ions at m/z 449. D, E, Localization patterns of the fragment ions at m/z 287 (E) and 317 (F), derived from the parent ions at m/z 449, corresponding to cyanidin-3-O-hexoside and petunidin-3-O-pentoside, respectively. F, Localization pattern of the ions at m/z 822 corresponding to PC (diacyl C36:3), which marks the nucellus epidermis/aleurone layer. G, Merged ion image of m/z 449→287 (red), m/z 449→317 (green) and m/z 822 (blue). H, Optical image of a black rice section after analysis of the ions at m/z 463. I, J, Localization patterns of the fragment ions at m/z 301 (I) and m/z 331 (J), derived from parent ions at m/z 463, corresponding to peonidin-3-O-hexoside and malvidin-3-O-pentoside, respectively. K, Localization pattern of the ions at m/z 822 corresponding to PC (diacyl C36:3). L, Merged ion image of m/z 463→301 (red), m/z 463→331 (green), and m/z 822 (blue). Scale bar: 1.0 mm.
Figure 5.
MALDI-IMS/MS analysis of ions at m/z 419 and m/z 449 on longitudinal or transverse sections of a black rice seed.
A–E, Longitudinal section images. Scale bar: 1.0 mm. F–J, Transverse section images. Upper side in transverse sections is the dorsal side of the black rice seed. Scale bar: 0.5 mm. A, F, Optical images. B, G, Localization pattern of the fragment ions at m/z 287, derived from the precursor ions at m/z 449, corresponding to cyanidin-3-O-hexoside. C, H, Localization pattern of the fragment ions at m/z 287, derived from the precursor ions at m/z 419, corresponding to cyanidin-3-O-pentoside. D, I, Localization pattern of the fragment ions at m/z 317, derived from the precursor ions at m/z 449, corresponding to petunidin-3-O-pentoside. E, J, Merged ion image of m/z 449→287 (red), m/z 419→287 (green), and m/z 449→317 (blue). K, Four regions defined in the intensity quantification of each fragment ion. L–N, Relative intensity of the fragment ions corresponding to cyanidin-3-O-hexoside (L), cyanidin-3-O-pentoside (M), and petunidin-3-O-pentoside (N). AV: anterior ventral region, PV: posterior ventral region, PD: posterior dorsal region, AD: anterior dorsal region. Data was collected from 7 sections of 3 black rice seeds (means ± S.E.). Different denote significant differences among groups means from a Tukey-Kramer test (p<0.01). O, HPLC chromatogram of anthocyanins in the black rice crude extract. Peak 1 and 2, unknown; peak 3, cyanidin-3-O-glucoside; peak 4, peonidin-3-O-glucoside. P, Q, Contents of cyanidin-3-O-glucoside (P) and peonidin-3-O-glucoside (Q) in each part of black rice seeds determined by HPLC and expressed in nmol per mg of seed pieces ± S.E in triplicate.