8.2.2 Enzymes Involved in Carbon
Metabolism: FEN1
The nitrogenase reaction requires 16 molecules of
ATP to reduce one molecule of dinitrogen gas.
This energy is produced by aerobic respiration of
bacteroides, using the carbon source supplied by
host plants. Carbon metabolism in host cells is
therefore critical to rhizobial symbiotic nitrogen
fixation. Indeed, the expression of genes encoding
enzymes involved in carbon catabolism is greatly
enhanced inL. japonicusnodules (Colebatch et al.
2004 ). One such gene, which encodes nodule-
enhanced phosphoenolpyruvate carboxylase, was
shown by RNA interference to be critical to rhi-
zobial symbiotic nitrogenfixation inL. japonicus
(Nomura et al. 2006 ). However, noL. japonicus
Fix−mutants defective in enzymes involved in
carbon metabolism have been isolated so far.
The analysis of another L. japonicus Fix−
mutant,fen1, revealed a novel metabolic reaction
in host plant cells crucial for rhizobial symbiotic
nitrogenfixation. The defective gene in thefen1
mutant was shown to encode homocitrate syn-
thase, which catalyzes the conversion of 2-oxo-
glutarate to homocitrate with acetyl-coenzyme A
(Imaizumi-Anraku et al. 1997 ; Kawaguchi et al.
2002 ; Hakoyama et al. 2009 ). Homocitrate is a
component of the FeMo-cofactor required for the
nitrogenase complex (Hoover et al. 1987 , 1989 ).
M. lotilacks theNifVgene encoding homocitrate
synthase (Hakoyama et al. 2009 ). This indicates
that host plant cells provide bacteroides with
homocitrate to support efficient nitrogen-fixation
activity. This hypothesis was supported by
results showing that the mutantfen1phenotype
was rescued either by expressing the wild-type
FEN1 gene or theAzotobacter NifVgene in
M. loti rhizobia, or by supplying synthetic
homocitrate. In Saccharomyces cerevisiae,
homocitrate is a precursor to the biosynthesis of
lysine. However, higher plants, including
legumes, are able to synthesize lysine from a
distinct pathway without homocitrate synthase.
Legumes are therefore likely to have acquired the
homocitrate synthase gene to overcome a lack of
theNifVgene in rhizobia. The identification of
theFEN1gene reveals a novel aspect of the
interrelationship between legumes and rhizobia
symbiosis, and further exploration could shed
light on the evolution of symbiosis.8.2.3 Proteins Expressed in Vascular
Tissues: SYP71Photosynthates assimilated by the host plant are
translocated from the shoots to the nodules, and
nitrogenfixed by rhizobia is transported from the
nodules to the shoots. The plant vascular system,
which carries both photosynthates and nitrogenous
compounds, contributes significantly to effective
symbiotic nitrogenfixation (as reviewed by Guinel
2009 ). Several genes required for nitrogenfixation
are expressed specifically in the vascular tissues of
nodules. Recently, a Fix−mutant defective in the
LjSYP71gene was identified (Hakoyama et al.
2012b).LjSYP71encodes a Qc-SNARE (soluble
N-ethylmaleimide sensitive factor attachment pro-
tein receptor) homologous toArabidopsis thaliana
SYP71. SNARE proteins are involved in vesicle
trafficking.LjSYP71was expressed in the whole
plant, and transcripts were detected in the vascular
tissues. The discovery of the LjSYP71-defective
Fix−mutant suggests the existence of a long-
distance signal transported as cargo in the vesicle,
which regulates nitrogen-fixing activity. It is well
known that the number of nodules that form on
legume roots is regulated by a long-distance signal
derived from the shoot (Oka-Kira and Kawaguchi
2006 ). Further efforts are required to clarify whether
the phenotype of theLjsyp71mutant is recovered
by shoot grafting and to determine what is trans-
ported in vesicle trafficking involving LjSYP71.8.2.4 Proteins Required
for Maintenance of Symbiosis:
IGN1The identification of the L. japonicus Fix−
mutantign1suggested that the host plant gene
was required for the maintenance of compatible
rhizobia in nodule cells (Kumagai et al. 2007 ).82 N. Suganuma