The 3′ untranslated region of the two cytosolic glutamine synthetase (GS1) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine

Abstract

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to produce glutamine. The GS enzyme is located either in the chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene family and the members exhibit differential expression pattern mostly attributed to transcriptional regulation. Based on our recent finding that a soybean GS(1) gene, Gmglnβ ( 1 ) is subject to its 3'UTR-mediated post-transcriptional regulation as a transgene in alfalfa (Medicago sativa) we have raised the question of whether the 3'UTR-mediated transcript destabilization is a more universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes, MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these transformants suggests that while both the 3'UTRs promote transcript turnover, the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs along with Gmglnβ ( 1 ) 3'UTR respond to nitrate as a trigger in transcript turnover. More detailed analysis points to glutamine rather than nitrate as the mediator of transcript turnover. Our data suggests that the 3'UTR-mediated regulation of GS(1) genes at the level of transcript turnover is probably universal and is used for fine-tuning the expression in keeping with the availability of the substrates.

The 5′ untranslated region of the soybean cytosolic glutamine synthetase β1 gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants

“…The data presented here, points to the 5′ UTR of the Gm glnβ 1 gene as the determinant of translation enhancement and confirms our earlier finding that the 3′UTR of the Gm glnβ 1 gene controls the turnover of the transcript (Ortega et al 2006; Simon and Sengupta-Gopalan 2010). In this study, a transient assay using agroinfiltration has allowed us to simultaneously check the expression of different gene constructs involving the 5′ and 3′UTRs of the Gm glnβ 1 gene at the level of transcript and protein accumulation.…”

“…We have found that regulation of expression of a soybean ( Glycine max ) GS 1 gene (Gm glnβ 1 ) driven by a constitutive promoter (CaMV 35S) in transgenic plants occurs at a post-transcriptional level (Ortega et al 2001, 2006). We have also shown that the post-transcriptional regulatory step of the Gm glnβ 1 and the GS 1 genes from alfalfa is, at the level of transcript turnover, mediated by their 3′UTR (Ortega et al 2006; Simon and Sengupta-Gopalan 2010). The goal of this study was to investigate if Gm glnβ 1 is also subject to other modes of regulation by testing the role of its 5′UTR in the regulation of gene expression.…”

“…To understand the biological significance of the translation enhancement of the GS gene by its 5′UTR, it will be important to determine if this regulatory step is mediated by signals involved in the coordination of C and N balance that control gene expression (Nunes-Nesi et al 2010). There are reports in the literature suggesting regulation of cytosolic GS at the post-transcriptional level (Ortega et al 2006: Simon and Sengupta-Gopalan 2010), at the posttranslational level by phosphorylation (Finnemann and Schjoerring 2000; Lima et al 2006), through interactions with the 14-3-3 proteins (Diaz et al 2011) and at the level of enzyme stability (Ortega et al 1999), all steps being regulated by the N status. It is thus possible that GS 1 may also be regulated at the translational level in response to the C/N status of the cells.…”

Characterization of eight cytosolic sHSP genes and their expression in Capsella bursa-pastoris

“…The 3'-UTR, located between the stop codon and the poly(A) tail, may possess a variety of regulatory mechanisms. The 3'-UTRs of the two cytosolic glutamine synthetase genes were characterized as regulators of transcript stability in response to glutamine in Medicago sativa (Simon and Sengupta-Gopalan 2010). Splicing and alternative 3' end processing of mRNAs mediate riboswitch control of gene expression in plants (Wachter et al 2007).…”

Comparison of alfalfa plants overexpressing glutamine synthetase with those overexpressing sucrose phosphate synthase demonstrates a signaling mechanism integrating carbon and nitrogen metabolism between the leaves and nodules

“…The regulation of GS 1 gene expression is not limited to transcription (Sengupta‐Gopalan & Ortega, ). It has been shown that GS 1 is regulated at the level of transcript stability, mediated by its 3′UTR (Ortega et al., ; Simon & Sengupta‐Gopalan, ) and at the translational level by the 5′UTR (Ortega, Wilson, & Sengupta‐Gopalan, ). Furthermore, GS is subject to extensive posttranslational modification like phosphorylation (Finnemann & Schjoerring, ; Lima, Seabra, Melo, Cullimore, & Carvalho, ), ubiquitination, and binding with other proteins (Seabra, Silva, & Carvalho, ).…”