In Vivo Chemical Analysis of Plant Sap from the Xylem and Single Parenchymal Cells by Capillary Microsampling Electrospray Ionization Mass Spectrometry.

Abstract

In plants, long-distance transport of chemicals from source to sink takes place through the transfer of sap inside complex trafficking systems. Access to this information provides insight into the physiological responses that result from the interactions between the organism and its environment. In vivo analysis offers minimal perturbation to the physiology of the organism, thus providing information that represents the native physiological state more accurately. Here we describe capillary microsampling with electrospray ionization mass spectrometry (ESI-MS) for the in vivo analysis of xylem sap directly from plants. Initially, fast MS profiling was performed by ESI from the whole sap exuding from wounds of living plants in their native environment. This sap, however, originated from the xylem and phloem and included the cytosol of damaged cells. Combining capillary microsampling with ESI-MS enabled targeted sampling of the xylem sap and single parenchymal cells in the pith, thereby differentiating their chemical compositions. With this method we analyzed soybean plants infected by nitrogen-fixing bacteria and uninfected plants to investigate the effects of symbiosis on chemical transport through the sap. Infected plants exhibited higher abundances for certain nitrogen-containing metabolites in their sap, namely allantoin, allantoic acid, hydroxymethylglutamate, and methylene glutamate, compared to uninfected plants. Using capillary microsampling, we localized these compounds to the xylem, which indicated their transport from the roots to the upper parts of the plant. Differences between metabolite levels in sap from the infected and uninfected plants indicated that the transport of nitrogen-containing and other metabolites is regulated depending on the source of nitrogen supply.