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
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.
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
(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
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.
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.