Abstract
Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants. Although the GSTs gene family has been reported in many land plants, our knowledge of the evolution and function of the willow GSTs is still limited. In this study, 22 full-length GST genes were cloned from Salix babylonica and divided into three classes based on the conserved domain analysis, phylogenetic tree and gene structure: tau, phi and DHAR. The tissue-specific expression patterns were substantially different among the tau and phi GSTs. The Salix GST proteins showed functional divergences in the substrate specificities, substrate activities and kinetic characteristics. The site-directed mutagenesis studies revealed that a single amino acid mutation (Ile/Val53→Thr53) resulted in the lowest activity of SbGSTU7 among the Salix GSTs. These results suggest that non-synonymous substitution of an amino acid at the putative glutathione-binding site may play an important role in the divergence of enzymatic functions of Salix GST family.
Highlights
Glutathione S-transferases (GSTs; EC 2.5.1.18) are encoded by a large gene family and widely distributed in both prokaryotes and eukaryotes
Based on the analysis by the conserved domain of the National Center for Biotechnology Information (NCBI), 22 putative Salix GST proteins were identified to belong to the GST classes of tau, phi or dehydroascorbate reductase (DHAR), and their coding sequences were successfully cloned from Salix babylonica (Table 1)
In order to further confirm the subfamily designations of these GSTs, the phylogenetic tree was constructed using 22 Salix and 81 Populus GSTs, which indicates that the tau, phi and DHAR GSTs of Salix were clustered with the corresponding classes of Populus GSTs with high bootstrap support (Figure 2)
Summary
Glutathione S-transferases (GSTs; EC 2.5.1.18) are encoded by a large gene family and widely distributed in both prokaryotes and eukaryotes. They are multifunction proteins whose functions include detoxification, since they mainly catalyze the detoxification of a series of xenobiotics by conjugating the reduced glutathione (GSH) to various hydrophobic and electrophilic compounds (Dirr et al, 1994; Armstrong, 1997). Based on the amino acid sequence similarity, gene structure and substrate specificity, the plant GSTs have been divided into eight classes, including phi, tau, theta, zeta, lambda, dehydroascorbate reductase (DHAR), tetrachlorohydroquinone dehalogenase (TCHQD) and γ-subunit of the eukaryotic translation elongation factor 1B (EF1Bg) (Edwards et al, 2000; Oakley, 2005; Lan et al, 2009). The GSTs classes of tau and phi are the most abundant in terrestrial plants among these ten GST classes, except in non-vascular plants that have no tau GSTs (Frova, 2006; Liu et al, 2013; Labrou et al, 2015); they were observed only in plants (Basantani and Srivastava, 2007) and are largely responsible for the detoxification processes
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