GS activity is found in many plant tissues and organs and is derived from two enzymes, GS1 and GS2. GS1 is an abundant cytosolic enzyme in vascular tissues of roots, aging leaves, and developing seeds. Equally abundant, GS2 is a plastidic enzyme in photosynthesizing leaves, in roots as well as in other tissues in a manner that varies between different plant species. Both GS1 and GS2 are encoded by small gene families (Ireland and Lea, 1999; Lam et al., 1995; Oliveira et al., 2001). The functions of the GS1 and GS2 gene families have been studied in a number of plant species by analysis of the spatial and temporal expression patterns of their genes as well by genetic approaches. These have been described and discussed in other reviews (Hirel and Lea, 2001; Ireland and Lea, 1999; Lam et al., 1995; Lea and Ireland, 1999) and therefore will not be discussed in detail. The major function of GS2 emerging from these studies is to reassimilate ammonium ions generated by photorespiration, although GS2 also participates in the assimilation of ammonium-derived moieties from soil nitrogen (Lam et al., 1995; Miflin and Habash, 2002). The major functions of GS1 are to assimilate ammonium ions into glutamine in roots, and in senescing leaves for nitrogen transport between source and sink tissues (Lam et al., 1995; Miflin and Habash, 2002).
Does the GS-catalyzed assimilation of ammonium ion into glutamine represent a limiting factor for nitrogen use efficiency and plant growth? If the answer to this question is yes, three additional questions arise: (1) Does the rate-limiting effect of GS result either from insufficient nitrogen assimilation and transport between sources and sinks, or from insufficient reassimilation of ammonium ion derived from photorespiration (a fact that can cause ammonium ion toxicity), or both? (2) Can GS1 compensate for the function of GS2 and vice versa? (3) Is GS activity rate limiting in all or only in specific plant organs and tissues? These questions have been addressed by the use of recombinant gene constructs expressing GS1 and GS2 enzymes from different plants in different transgenic species and by utilizing different promoters.
Most studies on GS overexpression utilized the strong constitutive 35S promoter from the Cauliflower mosaic virus (CaMV), which leads to ectopic expression of the gene in most plant tissues. Genes encoding cytosolic GS1 from different plant species have been expressed in various plant species, including legumes, tobacco, and even poplar trees (Eckes et al., 1989; Fei et al., 2003; Fuentes et al., 2001; Gallardo et al., 1999; Hirel et al., 1992; Lam et al., 1995; Oliveira et al., 2001, 2002; Ortega et al., 2001; Temple et al., 1993; Vincent et al., 1997). These studies resulted in variable results apparently due to differential posttranscriptional and posttranslational controls of GS expression (Finnemann and Schjoerring, 2000; Miflin and Habash, 2002; Moorhead et al., 1999; Ortega et al., 2001). However, in many cases, GS1 overexpression caused increases in plant growth, particularly under nitrogen-limiting conditions, in total protein as well as chlorophyll content and photosynthesis. In the case of transgenic tobacco expressing a pea GS1 gene, the improved growth was dependent on light, but not on nitrogen supplementation. This suggests that the overexpressed GS1 improved photorespiratory ammonium ion assimilation in photosynthetic tissues (Oliveira et al., 2002), a function generally attributed to GS2. This was supported by the fact that these transgenic tobaccos also exhibited increased levels of intermediate metabolites of the photorespiratory process, as well as an increased CO2 photorespiratory burst (Oliveira et al., 2002). Taken together, the ability of cytosolic GS1 to compensate for rate-limiting activities of the plastid-localized GS2 suggests that both ammonium ion and glutamine shuttle quite efficiently between the cytosol and the plastid. Indeed, the levels of free ammonium ion were significantly reduced in some of the transgenic plants implying that ammonium ions were more efficiently converted into glutamine.
In other studies, recombinant GS proteins were expressed in transgenic plants using nonconstitutive promoters. Expression of a soybean GS1 gene under the control of the putative root-specific rolD promoter in transgenic Lotus japonicus and transgenic pea plants resulted in reduced root ammonium ion levels as well as in reduced plant biomass (Fei et al., 2003; Limami et al., 1999). These interesting results suggest that the GS-catalyzed incorporation of ammonium ion into glutamine in the roots, although important for root metabolism, antagonizes plant growth. It also implies that, at least in L. japonicus and pea, transport of ammonium ion from roots to the shoots and its incorporation into glutamine in above ground tissues is a preferred route for efficient plant nitrogen use compared to the assimilation into glutamine in the roots.
In another study, a bean GS1 gene was expressed in wheat under control of the rbcS promoter (Habash et al., 2001; Miflin and Habash, 2002). This promoter is highly expressed in young photosynthetic leaves, but not in roots. Although the promoter is highly expressed in young leaves, GS activity in the transgenic plants was enhanced only late in development of flag leaves, similar to the developmental pattern observed for endogenous wheat GS activity (Habash et al., 2001; Miflin and Habash, 2002). This unanticipated pattern was explained by the possibility that expression of the transgenic pea GS gene was subject to post-translation control in wheat (by?) the foreign wheat host. Nevertheless, since GS activity in late wheat flag leaves is crucially involved in nitrogen transport to the developing seeds, this allowed the investigators to analyze whether GS activity also limited the incorporation of nitrogen into glutamine for source/sink nitrogen transport. Indeed, the transgenic wheat exhibited increased growth rate as well as earlier flowering and seed development than the control nontransformed plants (Habash et al., 2001; Miflin and Habash, 2002), supporting a rate-limiting role for cytosolic GS activity in plant nitrogen use efficiency and transport from source to sink tissues.
These studies suggest that increasing GS activity by genetic engineering may be an important tool to improve nitrogen use efficiency and crop productivity, particularly under conditions of limiting nitrogen availability. This supposition is also supported by marker-assisted genetic studies in various crop plants in which a significant correlation was found between a number of important agronomical traits, such as nitrogen status and yield, and GS activity (Hirel and Lea, 2001; Jiang and Gresshoff, 1997; Limami and De Vienne, 2001; Masclaux et al., 2000). The importance of the GS trait is not only in improving yield but also in reducing environmental damage as a result of crop overfertilization. Modern agriculture has been associated with a dramatic increase in nitrogen fertilization, much of which is not assimilated by the plants resulting in contamination of the environment (Lawlor et al., 2001; Miflin and Habash, 2002; Ter Steege et al., 2001).
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