Spatiotemporal Study of Galactolipid Biosynthesis in Duckweed with Mass Spectrometry Imaging and in vivo Isotope Labeling

Isotope labeling coupled with mass spectrometry imaging (MSI) presents a potent strategy for elucidating the dynamics of metabolism in cellular resolution, yet its application to plant systems is scarce. It has the potential to reveal the spatiotemporal dynamics in lipid biosynthesis during plant development. In this study, we explore its application to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization (MALDI) MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied after growing in 50% D2O media over fifteen-day time period. When they were partially labeled after five days, three distinct binomial isotopologue distributions were observed corresponding to the labeling of partial structural moieties: galactose only, galactose and a fatty acyl chain, and the entire molecule. The temporal change of the relative abundance of these distributions follows the expected linear pathway of galactolipid biosynthesis. Notably, their MS images revealed the localization of each isotopologue group to the old parent frond, the intermediate tissues, and the newly grown daughter fronds. Besides, two additional labeling experiments, 1) 13CO2 labeling and 2) backward labeling of completely 50% D2O labeled L. minor in H2O media, confirm the observations in forward labeling. Further, these experiments unveiled hidden isotopologue distributions indicative of membrane lipid restructuring. This study suggests the potential of isotope labeling with MSI to provide spatiotemporal details in lipid biosynthesis in plant development.