Large-scale gene-expression analysis has
revealed that numerousL. japonicusgenes are
expressed differentially during nodule develop-
ment (Colebatch et al. 2002 , 2004 ; Kouchi et al.
2004 ). Some of these genes have been put for-
ward as good candidates for the regulation of
symbiotic nitrogenfixation, as they are specifi-
cally expressed in nodule cells and closely
associated with the onset of the process. For
instance, the expression of leghemoglobin genes
is induced exclusively in infected nodule cells
and concurrently with the onset of nitrogen-
fixation activity. When leghemoglobin gene
expression is suppressed in nodule cells, rhizo-
bial symbiotic nitrogenfixation is lost (Ott et al.
2005 ), indicating that leghemoglobin genes are
essential for symbiotic nitrogenfixation. How-
ever, until now, only a few genes have been
proven by reverse genetics to be indispensable to
nitrogenfixation.
Fix−mutants are useful for identifying host
plant genes that regulate rhizobial symbiotic
nitrogenfixation. Fix−mutants with nodules that
are normally endocytosed by rhizobia, but that
exhibit little or no nitrogen-fixing activity, have
been isolated inPisum sativumandMedicago
sativa(Vance and Johnson 1983 ; Kneen et al.
1990 ; Tsyganov et al. 1998 ). However, it has
been difficult to identify the causal genes in Fix−
mutants because the genomes of these crop
legumes are large. Isolation of such Fix−mutants
has been succeeded in the model legume L.
japonicus(Schauser et al. 1998 ; Szczyglowski
et al. 1998 ; Kawaguchi et al. 2002 ), and progress
in genome sequencing ofL. japonicus(Sato et al.
2008 ) has greatly facilitated the identification of
genes responsible for the Fix−phenotype with
the forward genetic approach. To date,five such
genes have been identified by map-based cloning
inL. japonicus(Fig.8.1). This chapter describes
the possible roles and implications of these genes
in symbiotic nitrogenfixation. The reader is also
referred to recent related reviews (Kouchi et al.
2010 ; Kouchi 2011 ; Udvardi and Poole 2013 ).
8.2 Host Plant Genes Critical
to Nitrogen Fixation8.2.1 Transport Proteins Located
on the Symbiosome Membrane:
SST1 and SEN1Rhizobia endocytosed in nodules are surrounded
by a symbiosome membrane, which is thought to
be derived from the plasma membrane of infec-
ted cells. The exchange of metabolites mediated
by the symbiosome membrane is essential for the
maintenance of rhizobial nitrogen fixation.
The carbon sources required by bacteroides for
the nitrogenase reaction are provided by host
plant cells, andfixed nitrogen is transported from
bacteroides to host plant cells through the sym-
biosome membrane (Udvardi and Day 1997 ;
White et al. 2007 ). It therefore acts not only as a
physical barrier between the host plant cell and
the bacteroides, but also as a critical player in
nitrogenfixation.
Map-based cloning of the gene responsible for
theL. japonicusFix−mutant has identified a
sulfate transporter located in the symbiosome
membrane (Wienkoop and Saalbach 2003 ) that
is indispensable for nitrogen-fixation activity
(Schauser et al. 1998 ; Kawaguchi et al. 2002 ;
Krusell et al. 2005 ). A mutant deficient in the
symbiotic sulfate transporter SST1 formed nod-
ules that were normally endocytosed with rhizo-
bia, but that exhibited lower nitrogen-fixation
activity than the wild type. Nitrogenase is made
up of NifHDK proteins containing three metal
clusters, 4S-4Fe cluster, P-cluster, and FeMo-
cofactor, all of which contain sulfur. The SST1
protein plays a role in transporting sulfate to the
bacteroides from the host plant. Sulfate trans-
porters are also required for plant growth, because
sulfur is found in a few amino acids. However, the
SST1gene is expressed exclusively in nodules
(Krusell et al. 2005 ). Furthermore, thesst1mutant,
which has nodules that do not express SST1,
can grow when combined nitrogen is supplied80 N. Suganuma