Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest.

本研究采用基质辅助激光解吸电离质谱成像（MALDI mass spectrometry imaging）技术，对大麦萌发前七日的代谢物进行定位分析。实验共检测到至多100个质谱信号，其中85个信号被鉴定为48种具有显著组织特异性定位特征的代谢物。寡糖可在胚乳以及发育完全的胚的部分区域中被观测到。胚乳中的脂质会依据其脂肪酸组成发生共定位现象，且二酰基磷脂酰胆碱的分布会随萌发过程发生变化。研究在胚中检测到26种具有潜在抗真菌活性的大麦碱，其糖基化、羟基化及O-甲基化衍生物均呈现组织特异性定位特征。为揭示局部代谢物组成的时空分布模式，本研究对时序产生的多组质谱成像数据集进行了批量分析，该方案需采用全新的预处理策略以实现不同数据集间的可比性，同时还需配套开发适用于无监督聚类的新方法。针对每个时间点样本得到的空间分割结果可通过交互式聚类图谱进行可视化，从而实现对所有时间点的同步交互式探索。借助这一全新的分析方法与可视化工具，本研究发现了代谢物模式随萌发过程的动态变化，其定位精度可达单质谱信号级别。本研究是首个采用MALDI质谱成像技术对谷类萌发过程进行时序代谢物谱分析的研究，共鉴定出大量具有农学与工业应用价值的化合物峰，包括寡糖、脂质及抗真菌物质。这些代谢物的精细定位结果，以及用于组织原位代谢物谱绘制的质谱聚类分析结果，为理解植物萌发过程提供了关键信息，具有极高的科学研究价值。