Plants produce an extraordinary number of metabolites whose biological activity enables them to successfully cope with everchanging conditions of the environment. Production of these metabolites is occurring through multiple biosynthetic pathways, many of which are still to be discovered. The key for discovering these unknown biosynthetic pathways, as well as new functions of the already known enzymes, is identification of their metabolic products, many of which are lowly abundant intermediates. With development of technology, discovery and identification of new metabolites has advanced substantially in aspects of their detection, quantification and localization, enabling more detailed insight into metabolism of plants.

In this PhD work, I have used various plant species to study the presence and metabolism of glutathione conjugates. Through different means of induction, I investigated metabolism of glutathione conjugates and their biosynthetic origin. This was done from the point of their potential precursors and responsible biosynthetic enzymes, presumed to be glutathione transferases (GSTs).

Considering the current lack of knowledge on localization of glutathione conjugates (GS-conjugates), I aimed to develop a method that would enable their localization, helping to discover the link between GS-conjugates and GSTs responsible for their production. In order to achieve this, I used Matrix Assisted Laser Desorption and Ionization - Mass Spectrometry Imaging (MALDI-MSI). With low abundance of naturally present GS-conjugates and difficulty of controlled induction of their biosynthesis, I also decided to investigated localization of other general and specialized metabolites in plant tissue. Considering the challenges of using MALDI-MSI on plant tissues, my investigations led me to novel approach in the field of MSI, known as MALDI-2-MSI, which I explored as potential solution in investigation of lowly abundant plant metabolites such as glutathione conjugates.

Results of this thesis revealed that GS-conjugates are found in evolutionary distant plant species, both in normal physiological state of the plant and under biotic and abiotic stress. Induction of GS-conjugates biosynthesis was possible through mechanical stress and exposure to volatile organic compounds, although to which extend was this catalyzed by GSTs remained unclear.

Nevertheless, identification of GS-conjugate remains as one of key aspects for future research of plant GSTs. Both MALDI-1-MSI and MALDI-2-MSI were successfully used in localization studies of general and specialized metabolites across plant samples. Application of MALDI-2-MSI showed great potential for analysis of lowly abundant compounds, specifically phytohormones whose localization was not analyzed before in this manner. Flexibility of this technique from the point of sample preparation and MSI analysis allowed creating highly specific methods, optimized for detection of lowly abundant compounds in plant tissue.