Abstract
Glutathione S-transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of toxic xenobiotics and oxidatively produced compounds to reduced glutathione, which facilitates their metabolism, sequestration, or removal. We report here that soybean (Glycine max) root nodules contain at least 14 forms of GST, with GST9 being most prevalent, as measured by both real-time reverse transcription-polymerase chain reaction and identification of peptides in glutathione-affinity purified extracts. GST8 was prevalent in stems and uninfected roots, whereas GST2/10 prevailed in leaves. Purified, recombinant GSTs were shown to have wide-ranging kinetic properties, suggesting that the suite of GSTs could provide physiological flexibility to deal with numerous stresses. Levels of GST9 increased with aging, suggesting a role related to senescence. RNA interference studies of nodules on composite plants showed that a down-regulation of GST9 led to a decrease in nitrogenase (acetylene reduction) activity and an increase in oxidatively damaged proteins. These findings indicate that GSTs are abundant in nodules and likely function to provide antioxidant defenses that are critical to support nitrogen fixation.
Highlights
Glutathione S-transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of toxic xenobiotics and oxidatively produced compounds to reduced glutathione, which facilitates their metabolism, sequestration, or removal
Six different GSTs were expressed in E. coli to produce recombinant proteins that revealed a wide range of kinetic properties, suggesting that the range of GSTs may provide physiological flexibility to deal with various toxic products
RNA interference (RNAi) silencing of GST9 in nodules of composite soybean plants resulted in decreased GST9 transcript levels, decreased nitrogenase activity, and increased levels of oxidized protein products
Summary
Two-dimensional (2-D) gels of proteins recovered from GSH-affinity chromatography of nodule crude extracts were examined as a first screen to detect possible GSTs. The full list of peptides identified (Supplemental Table S1) includes high frequencies of Suc synthase, lipoxygenases, and leghemoglobins This is probably a reflection of the abundance of these proteins in nodules and the incomplete separation provided by one-step affinity chromatography rather than of some relationship to GSH metabolism. Nodules from CGT-5214 plants (CvMV promoter and GST9-specific RNAi) showed only 60% of the acetylene reduction activity of AKK 1467B (control) nodules. Real-time RT-PCR was used to compare transcript levels of key genes in nodules from composite plants (Fig. 7) These data are expressed in terms of the Oxidation Products in Nodules. Two common indicators of oxidative damage were used: thiobarbituric acid-reactive substances (TBARS), for lipid peroxidation, and Oxyblot, for protein oxida-
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