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

Abstract

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS 1 ) or in the chloroplast (GS 2 ). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS 1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS 1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22-24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.

Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco

“…The GS 1 transformants in our present study, while exhibiting slower growth rate in the early stages of development compared to the NT plants and the other transformants, showed an extended life span and delayed senescence, more so, under LN. The inconsistency that has been observed in the literature with regards to the outcome of overexpressing GS 1 has been attributed to the transgene, the recipient of the transgene, and the growth conditions, specifically the availability and nature of N nutrients (Eckes et al 1989;Temple et al 1993;Oliveira et al 2002;Ortega et al 2004;Kirby et al 2006). GS besides being regulated transcriptionally, is also subject to post-transcriptional (Ortega et al 2006) and post-translational regulation (Ortega et al 1999;Lima et al 2006;Seabra et al 2013), but how these regulatory steps cause prolonged life span, delayed senescence and late flowering, is not known.…”

“…Poplar plants transformed with a pine GS 1 gene, showed increased growth in height (Gallardo et al 1999). Enhanced expression of cytosolic GS 1 in Lotus corniculatus and L. japonicus, lead to early flowering and plant senescence (Vincent et al 1997;Ortega et al 2004). Fuentes et al (2001) showed that tobacco transformants with an alfalfa GS 1 gene driven by the CaMV 35S promoter exhibited improved performance over NT plants only when grown under nitrogen deficient conditions, but not under HN conditions, even though the transformants exhibited increased GS activity under both N regimens.…”

“…Compared to the NT plants, the GS 1 transformants grew for extended periods and exhibited delayed flowering when grown under both LN and HN. While there are many reports of overexpression of GS 1 in different plants in the literature, the outcomes are very varied (Eckes et al 1989;Temple et al 1993;Oliveira et al 2002;Ortega et al 2004;Kirby et al 2006;Pascual et al 2008). Poplar plants transformed with a pine GS 1 gene, showed increased growth in height (Gallardo et al 1999).…”

“…However, the outcome has been rather inconsistent (Temple et al 1993;Gallardo et al 1999;Fuentes et al 2001;Oliveira et al 2002, Carvalho et al 2003Fu et al 2003;Harrison et al 2003;Ortega et al 2001Ortega et al , 2004. The inconsistency has been attributed to the transgene, the plant in question and the conditions of growth.…”

Molecular cloning and characterization of a cytosolic glutamine synthetase gene, a fiber strength-associated gene in cotton

“…Increases in plant growth have been observed as a consequence of GS1 over-expression (Fuentes et al 2001;Oliveira et al 2002;Fu et al 2003). Correlations exist between the over-expression of GS1 and an increase in overall growth, fresh weight and protein content in Lotus japonicus (Ortega et al 2004) and poplar (Jing et al 2004). There are reports of successful anti-sense RNA technology-mediated down-regulation of GS1 in plants resulting in physiological or biochemical changes (Carvalho et al 2003;Harrison et al 2003).…”