System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by approximately 50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.

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.

Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. This symbiosis is mutually beneficial, with the bacteria providing a source of nitrogen to the host while the plant supplies carbon to the symbiont. Nodule development is a complex process that is tightly regulated in the host plant cell through networks of gene expression. In order to examine this regulation in detail, a library of quantitative reverse transcription-polymerase chain reaction primer sets was developed for a large number of soybean (Glycine max) putative regulatory genes available in the current expressed sequence tag collection. This library contained primers specific to soybean transcription factor genes as well as genes involved in chromatin modification and translational regulation. Using this library, we analyzed the expression of this gene set during nodule development. A large number of genes were found to be differentially expressed, especially at the later stages of nodule development when active nitrogen fixation was occurring. Expression of these putative regulatory genes was also analyzed in response to the addition of nitrate as a nitrogen source. This comparative analysis identified genes that may be specifically involved in nitrogen assimilation, metabolism, and the maintenance of active nodules. To address this possibility, the expression of one such candidate was studied in more detail by expressing in soybean roots promoter β-glucuronidase and green fluorescent protein fusions. This gene, named Control of Nodule Development (CND), encoded a Myb transcription factor gene. When the CND gene was silenced, nodulation was reduced. These results, associated with a strong expression of the CND gene in the vascular tissues, suggest a role for CND in controlling soybean nodulation.