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

Abstract

The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions

“…Starch determination was performed in the pellets from the ethanol extracts following removal of the supernatant for Suc determination as previously described (Aleman et al 2010;Seger et al 2015). The supernatant containing the Glc (C6 units) released by enzymatic hydrolysis was transferred to 1.5 mL tube and 50 mL of the supernatant was diluted to 250 lL with water, and 1 mL Anthrone reagent was added.…”

“…Total soluble protein extraction for both western analysis and SPS activity determination were performed as described (Aleman et al 2010;Seger et al 2015). Proteins were analyzed by SDS-PAGE in 12 % acrylamide (w/v) gels using a Mini-PROTEAN electrophoresis apparatus (BioRad, Hercules, CA).…”

“…Total GS activity was measured using the transferase assay (Ferguson and Sims 1971) as described by Seger et al (2015). Transferase units were calculated from a standard curve of c-glutamyl hydroxamate.…”

“…Enzyme activity SPS activity was assayed by quantifying the fructosyl moiety of sucrose using the Anthrone test (Seger et al 2015) and the activity is expressed as nmol Suc-P mg -1 protein min -1 . Total GS activity was measured using the transferase assay (Ferguson and Sims 1971) as described by Seger et al (2015).…”

“…Since Suc plays such a crucial role in the functioning of different processes that are key to plant growth (Grof et al 2007;Tognetti et al 2013;Ruan 2014), efforts have been made to increase SPS activity using transgenic approaches and the up-regulation of SPS activity has been shown to alter growth and development in many different plants (Micallef et al 1995;Laporte et al 1997;Laporte et al 1997Laporte et al , 2001Nguyen-Quoc and Foyer 2001;Baxter et al 2003;Haigler et al 2007;Ishimaru et al 2008;Park et al 2008Park et al , 2009Coleman et al 2010;Tian et al 2010;Seger et al 2015;Maloney et al 2015).…”