9.3 Cytokinin
Cytokinins regulate cell division, induction of
shoot formation from callus, activation of lateral
bud growth, suppression of senescence, and
movement of nutrients. Lohar et al. ( 2004 )
investigated the cytokinin distribution in trans-
genicL. japonicuscontaining a chimeric gene
consisting of the promoter from theArabidopsis
response regulator geneARR5driving theGUS
reporter gene. Lohar et al. ( 2004 ) found that GUS
distribution in uninoculated L. japonicus was
similar to that inA. thaliana. In plants inoculated
withMesorhizobium loti, ARR5 expression was
observed in curled/deformed root hairs and in
nodule primordia, indicating that cytokinin had
accumulated in those cells. Several reports have
indicated that cytokinin promotes the root nod-
ulation process. For example, Sinorhizobium
melilotistrains that lacked the ability to form root
nodules because of a mutation in thenodAor
nodBgenes recovered this ability when trans-
formed with a plasmid containing theIPTgene,
which encodes cytokinin synthetase. This result
was very interesting because the phenotype was
complemented not by an intact version of the
mutated gene (i.e., nodAor nodB), but by a
cytokinin synthetase gene (Cooper and Long
1994 ). Cytokinin treatment induced root
nodule–like structures in uninoculated wild-type
L. japonicusand also inducedNINexpression
(Heckmann et al. 2011 ). Root nodule formation
was suppressed inL. japonicustransformed with
a chimeric gene consisting of the CaMV35S
promoter fused to eitherArabidopsis CKX3or
maizeCKX1, both of which are cytokinin deg-
radation genes (Lohar et al. 2004 ). Several years
later, Murray et al. ( 2007 ) and Tirichine et al.
( 2007 ) reported that a cytokinin signal was nec-
essary for root nodule formation inL. japonicus.
Genetic integration of the cytokinin phosphore-
lay pathway in root nodule formation has been
demonstrated using gain- and loss-of-function
mutants ofLjLHK1. In thehit1mutant (loss-of-
function), infection frequency was increased but
root nodules did not form (Murray et al. 2007 ).
Meanwhile, spontaneous root nodule formation
was observed in the snf2 (gain-of-function)
mutant, in which cytokinin signaling is consti-
tutively“on”(Tirichine et al. 2007 ). These facts
suggest that activation of cytokinin signaling is
involved in the induction of root nodule forma-
tion (Fig.9.1). In M. truncatula, root nodule
primordium formation was inhibited by RNAi-
mediated down-regulation of the gene for type A
cytokinin response regulator MtRR9 and
enhanced by its overexpression. Similar results
were observed inL. japonicustransformed with
MtRR9. Furthermore, the expression of L.Fig. 9.1 Effects of
phytohormones on root
nodule formation. Auxin
and cytokinin are required
for cortical cell division
and auxin acts downstream
of cytokinin signaling.
Ethylene, gibberellin, and
abscisic acid inhibit the
cortical cell divisions and
rhizobial infection
9 Hormone Regulation of Root Nodule Formation in Lotus 87