and 6. At least seven gene clusters were identified
that co-localize to the same contigs with paralo-
gous genes and short intergenic regions (e.g.,
2,032 bp between LjNPF5.3 and LjNPF5.4
genes) (Criscuolo et al. 2012 ).
The assignment of the 39L. japonicuscom-
plete members to the eight clades of the NPF
superfamily identified by Leran et al. ( 2014 ) and
their relative position within these was based on a
BLAST analysis where each of theL. japonicus
proteins were queried against the already assigned
members of theA. thalianaandM. truncatula
families. A nomenclature for the provisional list of
complete LjNPF members is indicated in
Tables12.1and12.2.L. japonicusNPF members
are distributed between all the eight subfamilies
with a peak of eight and a minimum of two
members in clades six and one, respectively.
Transcription ofNPF1andNRT2plant genes
is reported to be regulated by nitrate, nitrite,
ammonium, glutamine, N starvation, light,
sucrose, diurnal rhythm, and/or pH (Wang et al.
2012 ), and in some cases, the transcriptional
regulation is linked to a modulation of the nitrate
uptake activities. In the molecular characteriza-
tion reported by Criscuolo et al. ( 2012 ), a tran-
scriptional analysis of a subset ofL. japonicus
NPF and NRT2 genes showed a repressible,
inducible, and constitutive response to provision
of nitrate, auxin, or cytokinin. This analysis also
allowed the identification ofL. japonicus NPF
andNRT2genes induced during the symbiotic
interaction with M. loti with two genes,
LjNPF6.5/chr2.CM0826.370 and chr1.
CM0001.20, that were specifically expressed in
nodular tissue (Criscuolo et al. 2012 ). The pat-
tern of expression of theL. japonicus NPFand
NRT2 genes can be integrated by the large
amount of data reported in genome-wide analysis
comparing expression profiles in inoculated and
un-inoculated plants (Colebatch et al. 2004 ;
Kouchi et al. 2004 ;Høgslund et al. 2009 ). In
particular, we queried the large set ofL. japoni-
cus transcriptome data encompassing different
organs, stages of the symbiotic interaction, and
root nodules development in wild-type and
mutant genotypes (Høgslund et al. 2009 ). A large
number of Lotus NPF and NRT2 genes are
identified by the probe sets exploited in this
GeneChip approach, and the profiles of expres-
sion in roots and young and mature nodules are
reported in Table12.3(Høgslund et al. 2009 ).
Interestingly, seven members of the family that
do not include theLjNPF6.5cited above show a
clear-cut induction profile in nodular tissue.
Moreover, the level of expression of these nod-
ule-induced genes is not dependent on nitrogen
fixation as it is not affected in thefix-nodules
obtained in the sen1 and sst1 genetic back-
grounds (Høgslund et al. 2009 ). These data have
been further updated by the analysis reported by
Takahashi et al. ( 2012 ) that indicate the infection
zone as the nodule region where a peak of tran-
scription level is detected forfive out of the eight
NPF nodule-induced genes (Table12.3).
The subclassification of theL. japonicusNPF
members shown in Table12.1indicates that the
eight Lotus proteins preferentially expressed in
nodular tissue are distributed infive out of the
eight phylogenetic subfamilies identified by Leran
et al. ( 2014 ) with a peak of three nodule-induced
NPF genes in the clade 5. This distribution sug-
gests different biochemical and physiological
roles. Interestingly, the subfamily six with the
LjNPF6.2 and LjNPF6.5 proteins also includes the
A. glutinosaAgDCAT1 protein involved in the
supply of intracellular bacteria with dycarboxy-
lates (malate) in the actinorhizal/frankia symbiotic
interaction (Table12.1) (Jeong et al. 2004 ).
The molecular characterization of the L.
japonicus NRT2family allowed the identification
of a single gene (chr1.CM0001.20) that was
strongly induced in young and mature nodular
tissue (Criscuolo et al. 2012 ). In Table12.3, the
expression profiles of the LjNRT2.1 and
LjNRT2.2genes exported from the data reported
by Høgslund et al. ( 2009 ) are also indicated,
showing a significant down-regulation in young
and mature nodules. This pattern is consistent
with the one published forLjNRT2.1in Criscuolo
et al. ( 2012 ), while forLjNRT2.2,only a slight
decrease of the transcript level was reported.12 Nitrate Transport and Signaling 131