Constitutive Overexpression of Cytosolic Glutamine Synthetase (GS1) Gene in Transgenic Alfalfa Demonstrates That GS1 May Be Regulated at the Level of RNA Stability and Protein Turnover

Abstract

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH 4 ϩ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS 1 ) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of ␤-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-␤-glucuronidase gene construct, suggesting that the transcript for the GS 1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS 1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS 1 transcripts when fed with high levels of NO 3 Ϫ . However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS 1 is regulated at the level of RNA stability and protein turnover.

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

“…While there are several reports in the literature where an increase in plant growth has been observed as a consequence of GS 1 over-expression and the resulting increase in GS activity (Fuentes et al 2001;Gallardo et al 1999;Fu et al 2003;Oliveira et al 2002;Su et al 1995), there are cases where the presence of a GS 1 transgene is not accompanied by an increase in GS activity (Ortega et al 2001;Fei et al 2003). Since the GS 1 transgene, in most of these studies, is being driven by the constitutive CaMV35S promoter, it would further appear that the regulation of the transgene is at a step(s) beyond transcription.…”

“…Alfalfa plants transformed with a GS 1 gene driven by the CaMV 35S promoter showed a complete absence of the transgene transcript in the nodules (Ortega et al 2001). To investigate if the Ms glnβ transgene was expressed in the nodules of transformed L. japonicus and to demonstrate that the increase in GS activity and GS 1 protein in the M-4 and K-25 progeny was the result of the expression of the Ms glnβ transgene, total RNA was isolated from leaves, roots and nodules of control and transformed L. japonicus plants and following fractionation it was subjected to northern blot hybridization using the 3′-UTR of the Ms glnβ cDNA as the probe.…”

“…-(B) Two-dimensional SDS-PAGE was performed as previously described (Ortega et al 2001). An amount of protein equivalent to 0.05 µmol min −1 of transferase activity was loaded per sample.…”

“…Pea plants transformed with a soybean GS 1 gene driven by the CaMV 35S promoter or a nodule-specific promoter, while showing increased levels of GS 1 , showed no increase in GS activity or in total nitrogen or protein content. Similarly, our attempts to over-express alfalfa and soybean GS 1 genes in alfalfa in both a constitutive and nodule-specific manner have to date resulted in no increase in either the GS 1 polypeptide level or GS activity (Ortega et al 2001). There are some reports of successful antisense RNA technology-mediated down-regulation of GS 1 in plants resulting in physiological and/or biochemical changes (Temple et al 1994;Carvahlo et al 2003;Harrison et al 2003).…”

“…Several attempts have been made to modulate the levels of the GS enzyme in different plants using genetic engineering tools, with rather mixed outcomes (Eckes et al 1989;Hemon et al 1990;Hirel et al 1992Hirel et al , 1997Temple et al 1993Temple et al , 1994Temple et al , 1998Su et al 1995;Vincent et al 1997;Brugiere et al 1999;Gallardo et al 1999;Limami et al 1999;Ortega et al 2001;Fuentes et al 2001;Oliveira et al 2002;Carvalho et al 2003;Fei et al 2003;Fu et al 2003;Harrison et al 2003). Tobacco plants transformed with different GS 1 genes driven by the CaMV 35S promoter have shown an increase in GS activity and GS 1 polypeptide level in the leaves (Eckes et al 1989;Hemon et al 1990;Hirel et al 1992Hirel et al , 1997Temple et al 1993;Fuentes et al 2001;Oliveira et al 2002).…”