Decoding the function of Tau glutathione S-transferases GSTU24 and GSTU25 in Arabidopsis

• Applicant: Dr. José Manuel Ugalde
• Subject Area: Plant Physiology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 520823775

Glutathione S-transferases (GSTs) are ubiquitous enzymes with multiple functions, mainly linked to the detoxification of xenobiotic compounds and the removal of oxidized molecules. Plant GST families are extensive and commonly arrayed in tandem genes or large gene clusters in the genome due to gene duplication. This increases the chance of functional redundancy and makes their individual characterization highly challenging. Arabidopsis thaliana has 53 GSTs divided into 7 classes of which the plant-specific class tau (GSTU) with 28 members is the largest. With few exceptions GSTUs are organized in three gene clusters. Based on in vitro characterization, GSTUs have two GSH-dependent enzymatic activities: GSH-binding to the electrophilic center of potentially toxic compounds (GST activity) and a GSH-dependent peroxidase function towards lipid peroxides (GPOX activity). Additionally, they have a non-enzymatic ligandin function for binding of fatty acids. Among all GSTUs, GSTU24 and GSTU25 stand out with the highest GST and GPOX activities surpassing other GSTUs by a factor of 1000. GSTU24, GSTU25 and GSTU26 are arranged in a gene cluster named cluster 1. In this cluster GSTU24 and GSTU25 are the most highly induced genes under biotic and abiotic stress compared to other stress-related genes, suggesting a potential role in stress defense. Yet there is no distinct phenotype reported in null mutants for these genes. We have isolated a mutant line for GSTU25, which shows accelerated growth and increased resistance towards methyl viologen (MV)-induced oxidative stress due to reciprocal genetic compensation through upregulation of other members of cluster 1. This proposal aims to dissect the genetic redundancy of the cluster 1 GSTUs in Arabidopsis, characterizing the highly redundant GSTU24 and GSTU25 and decoding their critical functions under oxidative stress. As a strategy, we will generate variants of GSTU24 and GSTU25 deficient in their GSH-dependent functions. Wild-type and mutated GSTU variants will be tested for their ability to complement a yeast strain deficient in GSH-transferase activity and an Arabidopsis mutant lacking the transcription factors TGA2, TGA5 and TGA6 (tga256), which is known to be limited in the expression of 12 GSTUs, including all members of cluster 1. All complemented lines will be assayed for their tolerance toward MV-induced oxidative stress. Stress tolerance will also be tested in the double mutant gstu24 gstu25 and a line mutant for all cluster 1 GSTUs generated by CRISPR/Cas9. All cluster 1 GSTs are cytosolic and their mobility is likely important for their function. Therefore, we aim to evaluate the spacial relevance of GSTU24 and GSTU25 for performing their function by complementing the tga256 line with mis-targeted of GSTU24 and GSTU25 to other subcellular compartments or the plasma membrane. With these experimental strategies we expect to further dissect the individual roles of the highly redundant GSTs.

• DFG Programme: Research Grants