Glutathione conjugates are compounds generated in a reaction between the tripeptide glutathione (GSH) and reactive organic species. The reaction can be catalyzed by glutathione-transferases (GSTs), a group of enzymes that are highly abundant throughout the plant kingdom. In humans, GSTs detoxify xenobiotic compounds. However, with four times as many GST enzymes in plants compared to humans, many plant specific classes, and only few identified endogenous conjugates it is still an open question what is the reason for the abundance of GSTs in plants. The main objective of this work is to establish a functional imaging system for investigating the localization of glutathione conjugates in barley leaves, to understand their metabolism and aid the discovery of associated GSTs. Young Barley plants were treated with trans-2-hexenal and trans-2-octenal in different fumigation experiments. Plant extracts were analyzed by LC-qTOF-MS/MS and compounds identified by their retention times and MS/MS patterns compared to chemically synthesized authentic standards. Protein extracts from barley leaves were used in assays to determine whether the conjugation reactions were chemical or performed by GST enzymes. Leaf cross-sections of 10 µm thickness prepared by cryosectioning were freeze-dried and coated in DAN/DHB matrix by sublimation. Samples were then analyzed with 5µm resolution on the SMALDI-5 QExactive Orbitrap-MS system in both positive and negative polarity with 2D line mode and mass range 60-900 m/z. Presence of the glutathione conjugates of trans-2-hexenal (GS-3-hexanal) and trans-2-octenal (GS-3-octanal) in the fumigated leaves was confirmed in the LC-MS analyses. GS-3-hexanal was present in considerably greater amounts compared to the GS-3-octenal, pointing towards different substrate specificities of the responsible GSTs. The localization experiments are currently being performed and will demonstrate which matrix is most suitable for detection of GSH conjugates in plant tissues, and in which cell layers the GSTs responsible for their production can be found.

Localization of glutathione conjugates in barley leaves and seeds: Implications on potential metabolism

ACS Spring 2022

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