Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates.

Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine-specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes.

Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa.

Dehydroascorbate reductase (DHAR) is a key enzyme in the ascorbate-glutathione cycle that maintains reduced pools of ascorbic acid and serves as an important antioxidant. In this study, to investigate functional divergence of plant DHAR family and catalytic characteristics of the glutathione binding site (G-site) residues of DHAR proteins, we cloned three DHAR genes (PtoDHAR1/2/3) from Populus tomentosa and predicted the G-site residues. PtoDHAR1 protein was localized in chloroplast, while PtoDHAR2/3 proteins showed cytosolic localizations. Three DHAR proteins showed different enzymatic activities, apparent kinetic characteristics, optimum T m and pH profiles, indicating their functional divergence. Cys20, Lys8, Pro61, Asp72 and Ser73 of PtoDHAR2 were predicted as G-site residues based on their N-terminal amino acid sequence identity and the available crystal structures of glutathione S-transferases. The biochemical functions of these residues are examined in this study through site-directed mutagenesis. The aforementioned five residues are critical components of active sites that contribute to the enzyme's catalytic activity. Cys20, Pro61 and Asp72 of PtoDHAR2 are also responsible for maintaining proper protein structure. This study provides new insights into the functional divergence of the plant DHAR family and biochemical properties of the G-site residues in plant DHAR proteins.