japonicus LjRR6, which showed high similarity
toMtRR9, was increased by treatment with Nod
factor. These results further illustrate the
involvement of cytokinin signaling in root nod-
ule formation (Op den Camp et al. 2011 )
(Fig.9.1).
9.4 Ethylene
Ethylene is a gaseous phytohormone involved in
fruit ripening, leaf and fruit abscission, germi-
nation, seedling morphogenesis, root emergence,
root hair elongation, promotion offlowering,
senescence, and stress response. Many reports
have described the inhibitory effects of ethylene
in the root nodule formation process. For exam-
ple, ethylene inhibited the elongation of infection
threads into the inner cortex ofP. sativumroots
(Lee and LaRue 1992 ), and calcium spiking (a
response to bacterial nodulation signals) did not
occur in ethylene-treated M. truncatula. The
causative gene for the ethylene-insensitive M.
truncatula sickle mutant is EIN2, which is
involved in ethylene signaling. This mutant
showed a hyperinfection phenotype following
rhizobial inoculation (Penmetsa and Cook 1997 ;
Oldroyd et al. 2001 ; Penmetsa et al. 2003 , 2008 ).
Ethylene also shows inhibitory effects in L.
japonicus. Root nodule formation was sup-
pressed by treatment with 1-aminocyclopropane-
1-carboxylic acid (ACC), a precursor of ethylene,
but enhanced by treatment with aminoethoxyvi-
nyl glycine (AVG), which inhibits the activity of
ACC synthetase (ACS), and silver thiosulfate
(STS), which inhibits ethylene perception (Nukui
et al. 2000 ). Nukui et al. ( 2004 ) produced
transgenic L. japonicus carrying the mutated
melon ethylene receptor geneCm-ERS1/H70A,
which confers ethylene insensitivity. When
inoculated with M. loti, the transgenic plants
showed markedly higher numbers of infection
threads and nodule primordia on their roots than
control plants did. In addition, NINtranscript
levels increased in the inoculated transgenic
plants as compared to wild-type plants. Similar
results were obtained usingL. japonicustrans-
formed with a dominant-negative ethylene
receptor mutant gene fromArabidopsis(Lohar
et al. 2009 ). Interestingly, the number of root
nodules was not increased in an ethylene-insen-
sitive mutant ofG. maxthat had a mutation in
ethylene-resistance 1 (ETR1) (Schmidt et al.
1999 ). Consistent with these results, the number
of root nodules formed inG. maxtreated with
ACC, AVG, or STS was unchanged (Nukui et al.
2000 ).
ACC deaminase catalyzes the degradation of
ACC into ammonium andα-ketobutyrate, thus
reducing ethylene levels (Honma and Shimom-
ura 1978 ). The ACC deaminase gene (acdS) has
been found in many rhizosphere bacteria (Honma
and Shimomura 1978 ; Grichko and Glick 2000 ),
includingM. lotiMAFF303099 (Kaneko et al.
2000 ). InM. loti,acdSwas found in the symbi-
osis island, and the enhancing effect of this gene
on nodulation ofL. japonicusroots was dem-
onstrated by using anM. loti acdSdisruption
mutant (Uchiumi et al. 2004 ). Furthermore, DNA
macroarray analysis showed that a clone con-
taining acdS (mlr5932) was up-regulated in
bacteroid cells. These studies also showed the
negative effect of ethylene on root nodule for-
mation (Fig.9.1).
Legumes control nodule numbers through the
systemic AON process (Gresshoff 2003 ).
Mutants in this circuit have a supernodulating
phenotype (Krusell et al. 2002 ; Nishimura et al.
2002 ). A causative gene is the CLAVATA1-
related LRR receptor kinaseHAR1. BecauseL.
japonicusAON mutants are still ethylene-sensi-
tive, and a double mutant ofLjhar1/LjETR1- 1
does not show an additive phenotype, the AON,
and ethylene pathways are independent (Gress-
hoff et al. 2009 ).9.5 Gibberellin (GA)Gibberellin (GA) regulates seed germination,
stem elongation,flower bud formation, and fruit
enlargement. The effects of gibberellin on root
nodulation inL. japonicushave been reported in
detail (Maekawa et al. 2009 ). Exogenous appli-
cation of a biologically active GA, GA 3 , inhib-
ited the formation of infection threads and88 A. Suzuki