Fig 1.
The barley germination process: Seedling development and sampling time points.
A) Time scheme of mini malting of the Optic barley at 16°C for the collection of samples. Arrows indicate sampling time points with their sample name. 0d: raw barley, S: steeping, G: germination day, K: kilned malt (K not used for MSI). W: water, A: air rest, K1: kilning at 45°C (7h), K2: kilning at 65°C (17 h). B) Growth of the barley seeds during malting. Barley (0d, T = 1), steeped barley (S1d, T = 2), three of the five time points during germination (G1d, G3d, G5d (T = 4,6,8)) and final kilned malt (K1d) are shown as representatives. Main seed organs and compartments are indicated at the raw barley seed.
Table 1.
Detected and identified compounds from MS imaging of barley seeds.
Fig 2.
Localization of oligosaccharides in barley during germination.
A) Cryo-sections of non-germinated (0d) and three day germinated (G3d) barley. B) Average mass spectrum of all MS acquired from germinated barley with [M+Na]+ (red) and [M+K]+ (blue) ions of oligosaccharides. *DP: degree of polymerization. C) Intensity heat maps of oligosaccharides with three, six, and nine hexoses in sodium [M+Na]+ and potassium [M+H]+ adducts in ungerminated barley (0d) and after three days of germination (G3d). MS intensities were normalized to the TIC of each mass spectrum; the highest relative intensity of all MS was set to 100%. The distributions of these compounds at all time points of the germination process are provided as S5 Fig.
Fig 3.
Localization and signal intensities of hydroxycinnamic acid derivatives and hordatines in germinating barley.
Top: Localization of p-coumaroylagmatine (CA) as representative for hydroxycinnamic acid amides and hordatine B as representative for hordatines that co-localized to hordatine B when occurring in the same modification state. Intensity maps depict the non-glycosylated (m/z 581), glycosylated (m/z 743), and disaccharide-modified form (m/z 905) at three time points during germination (0d: non-germinated barley, G3d: three days germinated, G5d: five days germination) in longitudinal and transversal section plane. Hordatines were not detected in cross sections in non-germinated barley. The last panel shows an overlay of the three modification forms. Ion intensities were normalized to the TIC, the highest relative intensity was set to 100%. Middle panel: Average mass spectra from annotated embryo measurement regions (right) in non-germinated (green), three days (blue) and five days (red) germinated barley. Bottom: Mass spectrum with indicated peaks of hydroxycinnamic acid amides as hordatine precursors (m/z 250–350) and of hordatine A, B, C, and D (D not detected, grey font), hydroxylated hordatines (-OH), and hexose-modified derivates (Hex / 2 Hex) at m/z 550–1000. CA: coumaroylagmatine, FA: feruloylagmatine, CA-OH / FA-OH: hydroxylated CA and FA.
Fig 4.
Unsupervised spatial segmentation of 93 m/z intensities obtained from MALDI MSI of a three days germinated barley seed (G3d).
A: Image scan with outlines for the labeled seed compartments. B: Cluster map with the main compartments of the seed, transferred from A. C: Cluster distance wheel with 32 clusters set for this analysis; the 7 peak profile clusters presented in D are indicated with boxes. D: Mass lists of representative clusters; the bar size is indicative for the signal intensities; m/z identifications are provided in Table 1. See https://ani.cebitec.uni-bielefeld.de/barleymsi for an interactive version of the results in the WHIDE tool.
Fig 5.
Unsupervised spatial segmentation of eight independent MALDI MSI analyses covering the first days of barley germination using WHIDE.
A: Image scans with outlines of labeled seed compartments. B: Cluster analysis of all mass spectra of the whole grain areas. C: Cluster analysis of all mass spectra of the embryo areas as annotated in A. D: Cluster analysis of all mass spectra of the endosperm as annotated in A. E: Effect of the cluster granularity (7, 21, 56 clusters) on mapping results, exemplarily shown for four days germinated barley (G4d) from analysis B. The visualized cluster, granularity was set to 7 in A, B, and C to assign clear cluster profiles (right panels). 93 m/z values were selected for spatial segmentation in all analyses. Their contribution to the distinct clusters is indicated by the bar size in the right panels; for m/z identification see Table 1. For an interactive exploration of the results in WHIDE see https://ani.cebitec.uni-bielefeld.de/barleymsi.