different code names (Table11.1). There is a high
level of expression of theLjGdh4gene in leaves,
roots, and nodules from this plant.
11.3.4 Asparagine Synthetase (AS)
Three genes for asparagine synthetase (LjAsn1,
LjAsn2,andLjAsn3) have been identified inL.
japonicus (also called, respectively, LjAS1,
LjAS2, and LjAS3).LjAsn1 is highly or very
highly expressed in mature leaves, roots, and
nodules.LjAsn2is very highly expressed in roots
and nodules and barely detectable in leaves. The
third gene (LjAsn3) is poorly expressed in the
three types of tissues (Table11.1). The molecular
cloning and characterization of AS from L.
japonicusin relation to the dynamics of aspara-
gine biosynthesis in N-sufficient conditions has
been reported previously (Waterhouse et al.
1996 ). Three genes encoding AS have been
identified in other plant species, such asA. tha-
liana, which appear to be regulated in different
manners (Lea et al. 2007 ). Although there is
considerable variation between plants in the
exact mechanisms involved in the regulation of
the expression of AS, there is an overall con-
sensus: The expression of one gene (often that
which is most highly expressed) is induced by a
reduction in soluble carbohydrate supply and in
some cases darkness, while a second gene is
more widely expressed but may be stimulated by
carbohydrate and light. An increased supply of
reduced nitrogen, either as ammonium or amino
acids, induces expression of AS genes (Lea et al.
2007 ). Analysis of the amino acid sequences of
plant’s ASs shows that the proteins contain glu-
tamine, aspartate, and AMP-binding sites and are
related to theE. coliasparagine synthetase ASB
glutamine-dependent enzymes (Lea et al. 2007 ).
11.3.5 Asparaginase (NSE)
Three differentNsegenes encoding for aspara-
ginase have been identified in L. japonicus,
which showed a different pattern of expression
among leaves, roots, and nodules of the plants
(Table11.1). TheLjNse1gene is by far the most
highly expressed one, particularly in leaves. Two
of the genes present in L. japonicus, named
LjNse1 and LjNse3, encode for different K+-
dependent versions of the asparaginase enzyme,
while a third one, namedLjNse2,corresponds to
aK+-independent isoform. All these enzymes
have a α 2 β 2 tetrameric quaternary structure,
where theαandβsubunits correspond, respec-
tively, to the N-terminal (20 kDa) or C-terminal
(17 kDa) domains arising from a single proteo-
lytic event of each precursor, which is encoded
by the differentNsegenes. Structural and kinetic
studies revealed the crucial importance of K+for
the higher enzymatic activity and stability as well
as lower Km for asparagine and proper orienta-
tion of asparagine substrate within the LjNSE1
enzyme molecule (Credali et al. 2011 ). It was
proposed that LjNSE1 must be the main enzyme
responsible for the utilization of asparagine in
L. japonicusplants, while the K+-independent
isoform LjNSE2 is probably a detoxifying
enzyme in charge of the release of isoaspartyl
peptides arising from proteolytic degradation of
post-translationally altered proteins. TILLING
mutants affected inLjNSE1asparaginase isoform
were recently used to demonstrate by reverse
genetics, the importance of this particular iso-
form in plant growth and seed production. In
fact, the level of both legumin and convicilin
seed storage proteins was affected in the mutants
(Credali et al. 2013 ). Nevertheless,nse1mutants
indicated that there was no apparent involvement
of NSE1 protein in nodulation. Interestingly,
these results illustrate a key difference between
L. japonicusandArabidopsiswhere asparaginase
activity seems to be dispensable; in fact, inser-
tional mutants fromArabidopsislacking of one
or both K+-dependent (AspGB1) and K+-inde-
pendent (AspGA1) asparaginases develop nor-
mally (Ivanov et al. 2011 ). This differential
behavior amongLotusandArabidopsisregarding
asparaginases was attributed to the fact that
asparagine only accounts for approximately 5 %
of the total amino acids in the phloem sap of
Brassicaceae, whereas it accounts for almost122 C.M. Pe ́rez-Delgado et al.