Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?

Abstract

A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L. japonicus via transformation with Agrobacterium tumefaciens. Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity. Three of these lines were selected and their T 1 progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions. Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots. The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content. Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g. asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L. japonicus. Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated.Nitrogen (N) is one of the major limiting factors for plant growth. However, an excessive external supply of N causes major problems in agriculture and the environment, mostly due to nitrate leaching into underground water (Benes et al., 1989), not only polluting the aquatic environment but also resulting in a high nitrate content in food and drinks (Moller et al., 1990). Paradoxically, dinitrogen is 80% of the atmosphere, but it can only be assimilated by prokaryotic symbiotic or free-living diazotrophic organisms possessing nitrogenase activity (Dilworth, 1974). In higher plants, nitrate and ammonium are the two major inorganic N compounds that can be directly assimilated (Beevers, 1976). They are provided either artificially by the external supply of fertilizers, bacterial nitrification, or atmospheric reduction of dinitrogen during lightning, or naturally through biological N fixation. Therefore, regulation of inorganic N assimilation and incorporation of inorganic N into organic matter is of major importance not only in maintaining a sustainable agriculture but also protecting the environment.Over the past few years, attention has been particularly focused on the enzyme Gln synthetase (GS; E.C. 6.3.1.2) because of its central role in N metabolism and its diverse metabolic and developmental regulation in different plant species and organs (Cren and Hirel, 1999). Two major isoforms exist for the GS enzyme: cytosolic GS, occurring in the cytoplasm of leaves and non photosynthetic organs, and chloroplastic GS, present only in the chloroplasts of photosynthetic tissues and the plastids of roots or etiolated plants. The relative proportions of the cytosolic and plastidial GS activity may vary within different organs of the same plant or within dif...

How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model

“…A complex amino-acid cycling system between plant cells and bacteroids might prevent the bacteroids from assimilating NH 4 + , and allow NH 4 + to be secreted for uptake by the plant 88,101,103,104 . Metabolite analysis and RNAi depletion experiments suggest that NH 4 + assimilation by plant cells occurs primarily through glutamine and asparagine synthetases [105][106][107] . In L. japonicus, RNAi depletion of the next enzyme in the nitrogen assimilation pathway, glutamate synthase, results in a reduction of nitrogenase activity and the growth of stunted plants 108 .…”

Biological Nitrogen Fixation

“…El primer grupo incluye varias especies de regiones templadas, entre estas Lupinus subcarnosus, Pisum sativum y Medicago sativa y el segundo varias especies tropicales, tales como Glycine max, Phaseolus vulgaris y Vigna unguiculata (Scott et al, 1976;Miflin y Habash, 2002;Harrison et al, 2003).…”

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

“…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).…”