Identifying how genes and their functions evolve after duplication is central to understanding gene family radiation. In this study, we systematically examined the functional diversification of the glutathione S-transferase (GST) gene family in Populus trichocarpa by integrating phylogeny, expression, substrate specificity, and enzyme kinetic data. GSTs are ubiquitous proteins in plants that play important roles in stress tolerance and detoxification metabolism. Genome annotation identified 81 GST genes in Populus that were divided into eight classes with distinct divergence in their evolutionary rate, gene structure, expression responses to abiotic stressors, and enzymatic properties of encoded proteins. In addition, when all the functional parameters were examined, clear divergence was observed within tandem clusters and between paralogous gene pairs, suggesting that subfunctionalization has taken place among duplicate genes. The two domains of GST proteins appear to have evolved under differential selective pressures. The C-terminal domain seems to have been subject to more relaxed functional constraints or divergent directional selection, which may have allowed rapid changes in substrate specificity, affinity, and activity, while maintaining the primary function of the enzyme. Our findings shed light on mechanisms that facilitate the retention of duplicate genes, which can result in a large gene family with a broad substrate spectrum and a wide range of reactivity toward different substrates.

Late embryogenesis abundant (LEA) proteins are a large and highly diverse gene family present in a wide range of plant species. LEAs are proposed to play a role in various stress tolerance responses. Our study represents the first-ever survey of LEA proteins and their encoding genes in a widely distributed pine (Pinus tabuliformis) in China. Twenty–three LEA genes were identified from the P. tabuliformis belonging to seven groups. Proteins with repeated motifs are an important feature specific to LEA groups. Ten of 23 pine LEA genes were selectively expressed in specific tissues and showed expression divergence within each group. In addition, we selected 13 genes representing each group and introduced theses genes into Escherichia coli to assess the protective function of PtaLEA under heat and salt stresses. Compared with control cells, the E. coli cells expressing PtaLEA fusion protein exhibited enhanced salt and heat resistance and viability, indicating the protein may play a protective role in cells under stress conditions. Furthermore, among these enhanced tolerance genes, a certain extent of function divergence appeared within a gene group as well as between gene groups, suggesting potential functional diversity of this gene family in conifers.

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.