Even at early stages of nodule development,ign1
nodules contain irregularly shaped and enlarged
symbiosomes similar to lytic vacuoles with
multiple bacteroides. Furthermore, as the nodules
develop, infectedign1mutant cells disintegrate
rapidly and the bacteroides appear to aggregate.
The premature senescence observed in ign1
nodules is more rapid than that in other Fix−
mutants, which generally show a phenotype of
premature senescence, possibly because they
lack nitrogen-fixation activity. The rapid disin-
tegration of the symbiosomes might therefore not
be due simply to a lack of nitrogen-fixation
activity. The responsible gene,IGN1, encodes a
novel protein containing ankyrin repeats that
serve as a domain for protein–protein interac-
tions. In addition, the IGN1 protein is targeted to
the plasma membrane of infected cells. IGN1
might function as a membrane-anchorage protein
that regulates the subcellular localization of other
proteins, or interacts with and regulates other
membrane proteins or transporters. The IGN1
gene is expressed constitutively in all organs of
L. japonicusplants, but theign1mutant shows
no growth abnormalities other than the symbiotic
defect. IGN1 might thus be required to prevent
host plant cells from inappropriately invoking
premature senescence or as a kind of defense
system against microsymbionts, thereby playing
a critical role in the differentiation and/or per-
sistence of bacteroides and symbiosomes
(Kumagai et al. 2007 ). Elucidating the exact
biochemical function of IGN1 could provide new
insights into plant–rhizobium interactions.
8.3 Future Perspectives
Symbiotic nitrogenfixation in nodules is thought
to be supported by host plants at various levels,
including structure, metabolism, transport, and
recognition. The identification of additional Fix−
mutants could shed light on the host plant regu-
lation of rhizobial nitrogenfixation. Some such
mutants are currently under investigation (Hossain
et al. 2006 ; Sandal et al. 2006 ), and our knowledge
of the host plant genes that are essential for
symbiotic nitrogen fixation is predicted to
improve substantially in the near future. Analysis
of thefen1mutant revealed that its phenotype was
recovered by inoculation withM. lotitransformed
with theFEN1orAzotobacter NifVgene. How-
ever, the transformedM. lotistill lacked nitrogen-
fixation activity under free-living conditions. This
implies that other essential factors for rhizobial
symbiotic nitrogenfixation are supplied by the
host plant. The identification of the genes critical
to symbiotic nitrogenfixation also might help to
clarify the co-evolution of legumes and rhizobia.Acknowledgments The author thanks Dr. F.C. Guinel
(Wilfrid Laurier University, Waterloo, Canada) for critical
reading of the manuscript.ReferencesColebatch G, Kloska S, Trevaskis B et al (2002) Novel
aspects of symbiotic nitrogenfixation uncovered by
transcript profiling with cDNA arrays. Mol Plant-
Microbe Interact 15:411– 420
Colebatch G, Desbrosses G, Ott T et al (2004) Global
changes in transcription orchestrate metabolic differ-
entiation during symbiotic nitrogenfixation inLotus
japonicus. Plant J 39:487– 512
Guinel FC (2009) Getting around the legume nodule: II.
Molecular biology ofits peripheral zone andapproaches
to study its vasculature. Botany 87:1139– 1166
Hakoyama T, Niimi K, Watanabe H et al (2009) Host
plant genome overcomes the lack of a bacterial gene
for symbiotic nitrogenfixation. Nature 462:514– 517
Hakoyama T, Niimi K, Yamamoto T et al (2012a) The
integral membrane protein SEN1 is required for
symbiotic nitrogenfixation inLotus japonicusnod-
ules. Plant Cell Physiol 53:225– 236
Hakoyama T, Oi R, Hazuma K et al (2012b) The SNARE
protein SYP71 expressed in vascular tissues is
involved in symbiotic nitrogen fixation in Lotus
japonicusnodules. Plant Physiol 160:897– 905
Hoover T, Robertson AD, Cerny RL et al (1987)
Identification of the V factor needed for synthesis of
the iron-molybdenum cofactor of nitrogenase as
homocitrate. Nature 329:855– 857
Hoover TR, Imperial J, Ludden PW et al (1989)
Homocitrate is a component of the iron-molybdenum
cofactor of nitrogenase. Biochemistry 28:2768– 2771
Hossain MS, Umehara Y, Kouchi H (2006) A novel
Fix−symbiotic mutant ofLotus japonicus,Ljsym105,
shows impaired development and premature deterio-
ration of nodule infected cells and symbiosomes. Mol
Plant-Microbe Interact 19:780– 7888 Lotus Genes Involved in Nodule Function and Nitrogen Fixation 83