9.2 Auxin
Auxin functions as a signal molecule that con-
trols germination and growth, flower bud for-
mation,flowering, and embryo formation and
development. In addition, auxin is involved in
responses to environmental stimuli such as light
and gravity (i.e., tropisms). The roles of auxin in
root nodule formation have been studied for
many years in leguminous plants that form
indeterminate-type nodules (those that continue
to produce new cells after initiation), such as
Pisum sativum,Medicago sativa, andMedicago
truncatula.
Thimann ( 1936 ) reported thatP. sativumroot
nodules contained auxin and that the auxin con-
tent increased during root nodule development.
The Medicago truncatula-like AUX1 (MtLUX)
gene, which is similar to the AUX1 gene of
Arabidopsis thaliana, was expressed in the
region of each organ where vasculature arises (i.
e., in the center of lateral roots and the peripheral
region of nodules). These results suggest that
auxin is required at the stage of primordia
development and differentiation of the vascula-
ture within the nodule (de Billy et al. 2001 ). The
effects of treatment with auxin polar transport
inhibitors 1-naphthylphthalamic acid (NPT) and
2,3,5-triindobenzoic acid (TIBA) and auxin
antagonist∝-(phenylethyl-2-one)-indole-3-acetic
acid (PEO-IAA) on root nodule formation were
investigated usingLotus japonicus, which pro-
duces determinate-type nodules (nodules that
stop producing new cells shortly after initiation).
Both nodule number and nodule development
were reduced, and formation of the lenticel,
which normally develops on the root surface and
originates from the root outer cortex, was also
inhibited by the treatment (Takanashi et al.
2011 ). The expression of a transgene containing
the auxin-responsive promoter fromGlycine max
GH3fused to aGUSreporter gene was analyzed
inL. japonicus roots, and GUS activity was
observed in the vascular tissues of nodules.
These results suggest that auxin plays an
important role in the development of nodule
vasculature regardless of nodule type (Pacios-
Bras et al. 2003 ; Takanashi et al. 2011 ). Though
an auxin signal was detected in the dividing outer
cortical cells during thefirst nodule cell divisions
inL. japonicus, GUS expression was not detec-
ted in those same cells in white clover (Trifolium
repens), which produces an indeterminate-type
nodule. Flavonoids inhibit auxin transport, and
formation of indeterminate-type nodules is
affected byflavonoids. For example, root nodule
development was inhibited on hairy roots ofM.
truncatula in which chalcone synthase(CHS)
gene expression was suppressed; these roots had
a lower concentration offlavonoids, so auxin
transport was not inhibited. In contrast,flavo-
noid-regulated auxin transport inhibition is not
crucial during root nodule formation inG. max,
which produces determinate nodules (Subrama-
nian et al. 2006 ). InL. japonicusroot nodules, no
inhibition of auxin transport was observed (Pa-
cios-Bras et al. 2003 ). These differences in auxin
distribution and transport inhibition between
determinate and indeterminate nodules have been
attributed to the difference in developmental
pattern of the two nodule types.
Recently, Suzaki et al. ( 2012 ) investigated
auxin distribution during root nodule develop-
ment by using transformedL. japonicuscarrying
the auxin-responsive DR5 promoter fused to a
GFPgene with a nuclear localization signal. The
accumulation of auxin in the dividing cortical
cells was positively regulated by NIN (nodule
inception, a key transcription factor in nodule
development) and was inhibited by a systemic
negative regulatory mechanism called autoregu-
lation of nodulation (AON). Moreover, auxin
accumulation was observed in uninoculated roots
of theL. japonicusmutantspontaneous nodule
formation 2(snf2), which has a gain-of-function
mutation in LHK1 (the putative cytokinin
receptor lotus histidine kinase 1). Therefore, it
appears that auxin is involved in the division of
cortical cells and acts downstream of cytokinin
signaling (Fig.9.1).86 A. Suzuki