nodules, and this inhibition was counteracted by
the application of Uniconazole-P, an inhibitor of
GA 3 biosynthesis. Moreover, Nod factor–-
induced root hair deformation was severely
blocked in the presence of GA 3. GA treatment
also suppressed nodule formation on the roots of
snf2mutant plants, spontaneous nodulation trig-
gered by a gain-of-function mutation of calcium/
calmodulin-dependent kinase (CCaMK) (snf1),
and cytokinin-dependent induction of NIN.
These results indicate that GA inhibits the nod-
ulation signaling pathway downstream of cyto-
kinin (Fig.9.1).
The F box–containing protein SLEEPY1
(SLY1) functions as a positive regulator in GA
signaling. In the presence of GA, SLY1 interacts
with negative regulators of GA signaling (e.g.,
DELLA domain–containing GRAS proteins),
leading to their degradation (McGinnis et al.
2003 ; Dill et al. 2004 ). InL. japonicusover-
expressing SLEEPY, root nodule formation was
inhibited in spite of normal root development
(Maekawa et al. 2009 ). Thela cry-sconstitutive
GA signaling mutants inP. sativumalso form
significantly fewer nodules than wild-type plants.
However, GA deficiency resulting from thena
mutation inP. sativumalso causes a reduction in
nodulation, indicating that some level of GA
signaling is required for normal root nodule
development (Ferguson et al. 2011 ).
9.6 Abscisic Acid (ABA)
Abscisic acid (ABA) is involved in various stress
responses, seed maturation, germination, and
stomatal closure. ABA shows negative effects on
root nodule formation. Exogenous application of
ABA inhibited root nodule formation inP. sati-
vum(Phillips 1971 ),G. max(Cho and Harper
1993 ; Bano and Harper 2002 ), L. japonicus
(Suzuki et al. 2004 ),T. repens (Suzuki et al.
2004 ), and M. truncatula(Ding et al. 2008 )
(Fig.9.1). ABA was also able to inhibit root
nodule formation in the hypernodulation mutant
NOD1- 3 ofG. max(Cho and Harper 1993 ). In
both, L. japonicus and M. truncatula, ABA
treatment inhibited infection thread formation in
the root hair (Suzuki et al. 2004 ; Nakatsukasa-
Akune et al. 2005 ; Ding et al. 2008 ) (Fig.9.1).
Moreover, Ding et al. ( 2008 ) found that inM.
truncatula, calcium spiking after Nod factor
perception was inhibited by the application of
ABA. Phillips ( 1971 ) observed that cell division
induced by cytokinin was arrested by ABA
treatment and postulated that ABA had an
inhibitory effect on cortical cell division induced
by cytokinin. Ding et al. ( 2008 ) proved that this
idea was correct. ABA treatment inhibited
spontaneous root nodule formation in thesnf2
mutant ofL. japonicus and cytokinin-induced
ENOD40gene expression in the cortex of wild-
typeM. truncatula. These results suggest that
both infection thread formation in root hairs and
induction of cortical cell division induced by
cytokinin are regulated by ABA.
Due to the importance of root nodule function,
methods to increase the number of root nodules
have been of interest for many years. Studies of
hypernodulation mutants are good examples of
this interest. However, because production of
many nodules is costly to the plant, growth is
drastically affected in such mutants (Nishimura
et al. 2002 ). Theenf1(enhancednitrogenfixation
1 ) mutant was isolated by screeningL. japonicus
seedlings for survival on an agar medium con-
taining 70μM ABA, indicating reduced sensi-
tivity to ABA. The number of nodules formed on
enf1roots was approximately 1.7 times that of
wild-type MG20. The low ABA sensitivity of the
enf1mutant was thought to result from its lower
endogenous ABA concentration (Tominaga et al.
2009 ). Enhanced root nodule formation was also
observed in transgenicM. truncatulacarrying a
dominant-negative allele ofabscisic acid insen-
sitive1 from Arabidopsis (Ding et al. 2008 ).
Taken together, the available data indicate that
root nodule formation relies on an exquisite
balance between cytokinin and ABA. Though the
regulation of nodulation by ABA resembles the
effects of the negative regulator ethylene,
experiments with the ethylene-insensitivesickle
mutant show that ABA and ethylene function
independently in the regulation of lateral root
initiation, nodulation, and Nod factor signal
transduction (Ding et al. 2008 ).9 Hormone Regulation of Root Nodule Formation in Lotus 89