Inhibition of Glutamine Synthetase by Phosphinothricin Leads to Transcriptome Reprograming in Root Nodules of Medicago truncatula

Abstract

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

Extreme specificity of NCR gene expression in Medicago truncatula

“…However, all the NCR genes without exception are reduced in expression by the senescence-inducing treatments, in agreement with a recent report [30]. The downregulation of NCR gene expression is induced very rapidly, within 4 hours of nitrate application [30] or 8 hours of phosphinothricin treatment (Additional file 2: Figure S5) and occurs before the degeneration of the symbiotic cells and their bacterial symbionts [29]. The down-regulation of the NCRs most likely reflects the shutdown of the symbiotic process.…”

“…Nodule senescence leads to a complete digestion of the bacteroids and the symbiotic host cells and is controlled by the transcriptional activation of a battery of genes which are involved the digestion of macromolecules and the remobilization of the liberated nutrients [28]. To test whether NCR genes are induced by senescence, we analyzed their expression in nitrate or herbicide induced senescent nodules [20, 29] (Figure 4; Additional file 2: Figure S5). Besides the NCR genes, 8 senescence marker genes were included in the analysis (Additional file 1: Table S1) [28].…”

“…(A) Scatter plot of the expression of 334 NCRs (blue) and 8 senescence marker genes (red) in 14 dpi nodules (x-axis) and 16 dpi nodules, which have been treated before harvest with nodule senescence-inducing nitrate for 2 days (y-axis) [20]. (B) Scatter plot of the expression of 334 NCRs (blue) and 8 senescence marker genes (red) in 20 dpi nodules (x-axis) and 20 dpi nodules, which were treated for 24 h with phosphinothricin before harvest (y-axis) [29]. (C) Scatter plot of the expression of 334 NCRs (blue) and 8 senescence marker genes (red) in hand-sectioned nodule sample III (x-axis) and in hand-sectioned nodule sample IV (y-axis).…”

Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses

“…As it has been shown that nodules formed by the hmp - mutant rhizobium suffer a premature senescence induced by NO (Cam et al, 2012), it is tempting to speculate that the NO-induced GS inhibition could be associated with this premature nodule senescence. This idea is supported by the finding that the application of the GS inhibitor phosphinothricin (PPT) to root nodules promotes nodule senescence (Seabra et al, 2012). …”

Nitric oxide‐releasing antibacterial albumin plastic for biomedical applications

“…NO plays a crucial role in many physiological and pathological processes, including platelet activation and adhesion, vasodilation, wound healing, and inflammation . Because NO is a natural antimicrobial agent, it has been incorporated into different medical grade polymers to not only improve the hemocompatibility but also prevent infection in blood‐contacting devices such as sensors, catheters, vascular grafts, extracorporeal circuits and in medical device coatings . Moreover, NO releasing polymers have not been reported to cause any hemocompatibility, biocompatibility, or long‐term storage stability issues …”