this plant (Credali et al. 2013 ). Asparaginase
(NSE, EC 3.5.1.1) catalyzes the hydrolysis of
asparagine to yield aspartate and ammonia as
follows:
AsparagineþH 2 O!
NSE
AspartateþNH 4 þThe ammonia released by the asparaginase
reaction has to be subsequently reassimilated by
GS (Lea et al. 2007 ).
11.3 Genes for Ammonium
Assimilation
11.3.1 Glutamine Synthetase (GS)
As in other plant species, a small multigene family
is responsible for GS enzymatic activity inL.
japonicus. Five gene sequences encoding for
cytosolic GS (also called GS1 or Gln1) and
another one for plastidic GS (also calledGS2or
Gln2) were found in the available databases. Two
of the cytosolic genes are expressed at high
(LjGln1.1) or very high (LjGln1.2) levels in
leaves, roots, and nodules from this plant
(Table 11.1). The level of expression of the
LjGln1.2gene is about threefold higher in nodules
than in roots or leaves, while in the case of the
LjGln1.1gene, the level of expression is lower in
nodules and leaves than in roots (results not
shown). The third gene (LjGln1.3) is expressed at
low levels in leaves and at very low levels in roots
and nodules. A fourth and afifth cytosolic gene
(LjGln1.4andLjGln1.5) are only expressed at low
(LjGln1.4) or very low (LjGln1.5) levels in leaves.
Cytosolic GS polypeptides are the most abundant
in both roots and nodules ofL. japonicusplants
(García-Calderón et al. 2012 ; Orea et al. 2002 ).
Previous studies inL. japonicushad indicated that
a high level of GS activity in the root is negatively
correlated with above-ground biomass (Limami
et al. 1999 ). On the other hand, lowering GS
activity in nodules results in an increase in fresh
weight in nodules, roots, and shoots (Harrison
et al. 2003 ). This change in biomass could be
explained by more efficient ammonium assimila-
tion in the nodules of transformed plants, as
indicated by a large increase in amino acids
(mostly asparagine) with a concomitant decrease
in carbohydrate content (Harrison et al. 2000 ).
Other results established that constitutive over-
expression of GS1 inLotusproduce higher amino
acid levels and soluble protein concentration,
higher chlorophyll content, and a higher biomass
accumulation in the transgenic plants (Ortega
et al. 2004 ), while overexpression in shoots may
accelerate plant development, leading to early
senescence and premature flowering when
plants are grown on an ammonium-rich medium
(Vincent et al. 1997 ). In addition, it was also noted
that overexpression of GS1 in reproductive organs
critically affects their development and might be a
reason for sterility ofL. japonicusplants (Suárez
et al. 2003 ). In other plant species, 2–5 functional
Gln1genes have been generally reported. Differ-
ent versions of these genes have been found to be
associated with different roles in primary ammo-
nium assimilation and other forms of N recycling
in response to nitrogen availability in the external
medium, plant nitrogen status, light/dark condi-
tions, or abiotic/biotic stressors (Bernard and
Habash 2009 ). Cytosolic GS assimilates ammo-
nium from the three major types of nitrogen-fixing
symbiotic associations involving plants and either
Rhizobium, actinomycetes such as Frankia,or
cyanobacteria. In some species, a specific cyto-
solic GS isoenzyme is induced in nitrogen-fixing
root nodules, whereas in others, cytosolic GS that
is already present is involved (Bernard and
Habash 2009 ).
Regarding Gln2, a single gene has been
detected inL. japonicus, which has very high
levels of expression either in leaves, roots, or
nodules (Table11.1). Nevertheless, the levels of
transcript detected in leaves were found to be
aboutfivefold higher than those present in roots or
nodules from this plant (results not shown). In
most plant species, plastidic GS (GS2) is exclu-
sively, or very predominantly, expressed in green
tissues. However, the presence of GS2 was clearly
demonstrated in non-photosynthetic tissues of
temperate legumes (García-Calderón et al. 2012 ).
InMedicago truncatula, a secondGln2gene was
recently shown to be exclusively expressed in
developing seeds (Seabra et al. 2010 ). Thefirst120 C.M. Pe ́rez-Delgado et al.