Glutathione S-transferase gene family analysis and enzymatic properties of three selenium-responsive members from...

Selenium is an essential trace element for plant growth and development; however, excessive selenium stress induces the accumulation of reactive oxygen species, leading to oxidative damage and inhibition of normal plant growth. Cardamine hupingshanensis is a selenium‑hyperaccumulating plant species endemic to China, exhibiting strong tolerance and high capacity for accumulation in high‑selenium environments. Glutathione S‑transferases (GSTs) are important enzymes involved in diverse metabolic processes and stress responses, yet systematic identification and functional characterization of the GST gene family in this species remain incomplete. In this study, a genome‑wide identification of the GST gene family in C. hupingshanensis was performed using bioinformatics approaches, and the phylogenetic relationships, gene structures, conserved motifs, physicochemical properties, and subcellular localizations were systematically analyzed. A total of 61 ChGST genes were identified and classified into eight subfamilies, with the Tau and Phi subfamilies being the most abundant. Three representative Tau subfamily genes (ChGSTU4-3, ChGSTU16-1, and ChGSTU24), which exhibit divergent tissue‑specific and selenium‑responsive expression patterns and belong to separate phylogenetic clades, were selected for subsequent functional characterization. Prokaryotic recombinant expression vectors were constructed via gene cloning, and the recombinant proteins were expressed in Escherichia coli and purified. The temperature and pH adaptability, metal ion responses, and enzymatic kinetic parameters of the fusion proteins were determined, and their antioxidant activities were evaluated using the disk diffusion method. In vitro enzymatic assays showed that all three fusion proteins exhibited high catalytic activity in the range of 30-50 °C and pH 7.0-8.0. Different metal ions exerted distinct regulatory effects on ChGST enzyme activity: Na⁺, K⁺, and Ca²⁺ had negligible effects, whereas the other tested ions showed various degrees of inhibition. The disk diffusion assay demonstrated that ChGSTU4-3, ChGSTU16-1, and ChGSTU24 protected bacterial cells from oxidative stress induced by hydrogen peroxide. This study delineates the basic characteristics and in vitro functions of the GST gene family in C. hupingshanensis, providing preliminary experimental evidence that may contribute to understanding the molecular basis of selenium tolerance in this species, as well as a theoretical reference for mining stress‑responsive genes and for the bioremediation of selenium‑contaminated environments.