RESEARCH ARTICLE 3997
Development 139, 3997-4006 (2012) doi:10.1242/dev.084079
© 2012. Published by The Company of Biologists Ltd
Positive and negative regulation of cortical cell division
during root nodule development in Lotus japonicus is
accompanied by auxin response
Takuya Suzaki1,2,*, Koji Yano1, Momoyo Ito1, Yosuke Umehara3, Norio Suganuma4 and
Masayoshi Kawaguchi1,2
SUMMARY
Nodulation is a form of de novo organogenesis that occurs mainly in legumes. During early nodule development, the host plant
root is infected by rhizobia that induce dedifferentiation of some cortical cells, which then proliferate to form the symbiotic root
nodule primordium. Two classic phytohormones, cytokinin and auxin, play essential roles in diverse aspects of cell proliferation and
differentiation. Although recent genetic studies have established how activation of cytokinin signaling is crucial to the control of
cortical cell differentiation, the physiological pathways through which auxin might act in nodule development are poorly
characterized. Here, we report the detailed patterns of auxin accumulation during nodule development in Lotus japonicus. Our
analyses showed that auxin predominantly accumulates in dividing cortical cells and that NODULE INCEPTION, a key transcription
factor in nodule development, positively regulates this accumulation. Additionally, we found that auxin accumulation is inhibited
by a systemic negative regulatory mechanism termed autoregulation of nodulation (AON). Analysis of the constitutive activation of
LjCLE-RS genes, which encode putative root-derived signals that function in AON, in combination with the determination of auxin
accumulation patterns in proliferating cortical cells, indicated that activation of LjCLE-RS genes blocks the progress of further cortical
cell division, probably through controlling auxin accumulation. Our data provide evidence for the existence of a novel fine-tuning
mechanism that controls nodule development in a cortical cell stage-dependent manner.
INTRODUCTION
Under appropriate environmental conditions, legumes can form
nodular structures on their roots. Within the nodules, host plants
can obtain a nitrogen source fixed by soil bacteria (rhizobia); in
turn, the plants provide a carbon source for the rhizobia. This
mutual interaction between plants and rhizobia is defined as root
nodule symbiosis. During nodule development, plants respond to
nodulation (Nod) factors produced by the rhizobia; perception of
these factors by receptor kinases triggers a signaling cascade in the
epidermis of the root. As a result, dedifferentiation of some of the
cortical cells is induced; these cells subsequently divide to form the
nodule primordia (Szczyglowski et al., 1998; Oldroyd and Downie,
2008; Oldroyd et al., 2011). During the course of nodule
development, rhizobia invade the dividing cortical cells via a
tubular structure called the infection thread (Murray, 2011).
A number of genes involved in the positive regulation of
nodulation have been identified through analysis of nodulationdeficient mutants. Additionally, it has been postulated that another
genetic mechanism, termed autoregulation of nodulation (AON),
negatively regulates nodulation to moderate the number of nodules
formed (Caetano-Anollés and Gresshoff, 1991; Oka-Kira and
1
Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Aichi
444-8585, Japan. 2Department of Basic Biology, School of Life Science, Graduate
University for Advanced Studies (SOKENDAI), Okazaki, Aichi 444-8585, Japan.
3
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
4
Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542,
Japan.
*Author for correspondence ([email protected])
Accepted 5 July 2012
Kawaguchi, 2006; Ferguson et al., 2010; Kouchi et al., 2010; Reid
et al., 2011b). The basis of AON is systemic long-distance
signaling between root and shoot. In Lotus japonicus,
HYPERNODULATION ABERRANT ROOT FORMATION 1
(HAR1), which encodes a leucine-rich repeat receptor-like kinase,
is hypothesized to function in shoots where it recognizes and
responds to root-derived signals involved in the negative regulation
of nodulation (Krusell et al., 2002; Nishimura et al., 2002). Among
the candidates for such signaling molecules in L. japonicus are
CLE-ROOT SIGNAL1 (LjCLE-RS1) and LjCLE-RS2 (Okamoto
et al., 2009). These two proteins belong to the CLE (CLAVATA
3/ESR) family of proteins that play significant roles as signaling
molecules in cell-to-cell communication during a range of plant
developmental processes including stem cell maintenance, vascular
patterning and embryo development (Fletcher et al., 1999;
Hirakawa et al., 2008; Stahl et al., 2009; Fiume and Fletcher,
2012). AON appears to have a conserved molecular mechanism
among leguminous species, as functional counterparts of HAR1
and LjCLE-RS1/2 have been identified in Medicago truncatula and
Glycine max (Searle et al., 2003; Schnabel et al., 2005; Mortier et
al., 2010; Reid et al., 2011a). However, little is known about the
site of AON action in nodule development.
It has been shown that the phytohormones cytokinin and auxin
play fundamental roles in the control of cell proliferation and
differentiation in many developmental regulatory processes. The
putative cytokinin receptors LOTUS HISTIDINE KINASE 1
(LHK1) in L. japonicus and CYTOKININ RESPONSE 1 (CRE1)
in M. truncatula are involved in nodulation (Gonzalez-Rizzo et al.,
2006; Murray et al., 2007; Tirichine et al., 2007). Nodule formation
is impaired in plants carrying loss-of-function mutations of these
genes, whereas nodule-like organs (termed spontaneous nodules)
DEVELOPMENT
KEY WORDS: Autoregulation of nodulation, Auxin, CLE, Cytokinin signaling, Lotus japonicus, Nodule development
3998 RESEARCH ARTICLE
MATERIALS AND METHODS
Plant materials
The Miyakojima MG-20 ecotype of L. japonicus was used in this study.
MG-20 plants were mutagenized with ethylmethane sulfonate (EMS) and
cyclops-6, nin-9 and har1-8 were isolated from M2 plants. Allelism tests
indicated that all three genes were new mutants of cyclops, nin and har1,
respectively. The site of mutation in each mutant is shown in
supplementary material Table S1. The snf2 mutant, which has an MG-20
background, has been reported previously (Miyazawa et al., 2010). The
DR5::GFP-NLS construct was introduced into the mutants by crossing with
transgenic MG-20 plants.
Constructs and transformation of L. japonicus
The primers used for PCR are listed in supplementary material Table S2.
To generate the DR5::GFP-NLS construct, a 2.8 kb fragment of the
DR5::gateway-cassette (GW) was excised from a vector kindly provided
by J. Lohmann (Heidelberg University, Germany) and inserted between the
SacI and KpnI sites of pCAMBIA1300. The translational fusion of GFPNLS in pDONR221 (Invitrogen), which was kindly provided by D. Weigel
(Max-Planck Institute for Developmental Biology, Tübingen, Germany),
was inserted downstream of DR5 using the LR recombination reaction. The
resulting plasmid was introduced into Agrobacterium tumefaciens strain
AGL1 and transformed into MG-20 as described previously (Nishimura et
al., 2002).
To obtain the pCYCLOPS::mCherry-NLS construct, GFP was removed
using XhoI from the binary vector pCYCLOPS::GW; p35S::GFP reported
previously (Yano et al., 2008). The translational fusion of mCherry-NLS in
pDONR221, which was provided by D. Weigel, was inserted downstream of
the CYCLOPS promoter by the LR recombination reaction. For constitutive
expression of NIN, the GFP in the pLjUBQ::GW; p35S::GFP binary vector
(Maekawa et al., 2008) was removed using XhoI, and PCR-amplified mKO2
[Medical and Biological Laboratories (Sakaue-Sawano et al., 2008)] was
inserted into the XhoI site to create the new binary vector pLjUBQ::GW;
p35S::mKO2. NIN cDNA was amplified by PCR and cloned into the
pENTR/D-TOPO vector (Invitrogen). The NIN cDNA in pENTR/D-TOPO
was inserted downstream of the LjUBQ promoter by the LR recombination
reaction. To create pLjUBQ::LjCLE-RS1; p35S::GFP-LjLTI6b or
pLjUBQ::LjCLE-RS2; p35S::GFP-LjLTI6b constructs, first, a GFP from
which the stop codon had been deleted was amplified by PCR and cloned
into a pENTR1A (Invitrogen)-based vector (pJL-Blue), which has a multicloning site between the attL1 and attL2 sequences; the plasmid was kindly
provided by D. Weigel. The resulting vector was named pENTR-GFP.
In order to identify a marker for the plasma membrane in L. japonicus
(GFP-LjLTI6b), we performed a BLAST search (Altschul et al., 1990) of the
L. japonicus genomic sequence database for the chr5.CM0048.40 gene using
as the query the amino acid sequence of Arabidopsis LTI6b, which encodes
a plasma membrane-localized protein (Reddy et al., 2004). We designated
the recovered gene sequence LjLTI6b. The LjLTI6b cDNA was amplified
with a short alanine linker sequence by PCR and inserted downstream of the
C-terminus of the GFP in pENTR-GFP to produce a translational fusion of
GFP-LjLTI6b. The resulting GFP-LjLTI6b was amplified by PCR and used
to replace the original GFP of the pLjUBQ::GW; p35S::GFP vector to form
the new binary vector pLjUBQ::GW; p35S::GFP-LjLTI6b. The LjCLE-RS1,
LjCLE-RS2 and GUS cDNAs in pEDONR221 (Okamoto et al., 2009) were
inserted downstream of the LjUBQ promoter using the LR recombination
reaction. The recombinant plasmids were introduced into A. rhizogenes strain
AR1193 and were transformed into roots of DR5::GFP-NLS plants by a
hairy root transformation method described previously (Okamoto et al.,
2009).
In situ hybridization
The primers used for PCR are listed in supplementary material Table S2.
The in situ hybridization probes for GFP and HISTONE H4 were created
by PCR amplification of 495 bp and 372 bp fragments from each,
respectively. The fragments were inserted into a pGEM-T Easy vector by
TA cloning (Promega). Probe synthesis, preparation of sections and in situ
hybridization were performed as described previously (Suzaki et al., 2004).
Signals were observed with a light microscope (BX-50, Olympus).
DEVELOPMENT
are formed in the absence of rhizobia in the spontaneous nodule
formation2 (snf2) mutant that has a gain-of function mutation of
LHK1. Exogenous application of cytokinin to L. japonicus roots
has been shown to induce the formation of spontaneous nodules
(Heckmann et al., 2011). Some response regulators, which are
known to be components of the cytokinin signaling pathway, are
reported to be involved in nodulation in both species (Op den
Camp et al., 2011). The various reports described above suggest
that activation of cytokinin signaling is a pivotal event in nodule
initiation. Downstream of cytokinin signaling, two putative
transcription factors, NODULE INCEPTION (NIN) and
NODULATION SIGNALING PATHWAY2 (NSP2) (Schauser et al.,
1999; Heckmann et al., 2006; Murakami et al., 2006), have been
suggested to be involved in nodule organogenesis on the basis that
spontaneous nodule formation in the snf2 mutant is suppressed if
either of these genes has also been mutated (Tirichine et al., 2007).
NIN is thought to be required for nodule organogenesis because
nodule formation is completely blocked in nin mutants (Schauser
et al., 1999). Upon rhizobial infection, expression of NIN is
strongly activated both in the epidermis and in the nascent nodule
primordium. Recently, it has also been found that constitutive
expression of NIN can induce ectopic cortical cell division in the
absence of rhizobia (T. Soyano and M. Hayashi, personal
communication). Thus, NIN appears to play a crucial role in the
dedifferentiation and subsequent proliferation of cortical cells
during nodule development.
In comparison to cytokinin, relatively little is known about the role
of auxin in nodule development. There have been some
physiological studies on auxin, mainly in the genus Medicago. More
than twenty years ago, for example, it was shown that inhibition of
polar auxin transport induces the formation of pseudonodules in the
absence of rhizobia in M. sativa (Hirsch et al., 1989), a finding
recently confirmed in M. truncatula (Rightmyer and Long, 2011).
Furthermore, it was reported that silencing of the flavonoid pathway,
which acts to inhibit auxin transport, causes a reduction in nodule
number (Wasson et al., 2006). These observations suggest that
localized accumulation of auxin at the sites of incipient nodule
primordia might be a key step in nodule development. In addition,
expression of some MtPIN genes is upregulated in the cre1 mutant
of M. truncatula, suggesting that one role of cytokinin signaling in
nodulation might be to establish local auxin accumulation through
control of expression of such auxin transporters (Plet et al., 2011).
Although some understanding of the behavior of auxin in nodulation
has been achieved, there is still considerable uncertainty as to how
and when auxin acts in the various genetic pathways that control
nodule development.
Here we identify the site of auxin action during nodule
development in L. japonicus. Our detailed analysis of the
spatiotemporal induction pattern of auxin shows that cortical cell
division occurs concurrently with strong induction of auxin
accumulation. We show that auxin accumulation is under the
positive regulation of cytokinin signaling and that NIN functions in
the local accumulation of auxin at cortical cells. Our results further
show that AON signaling, including HAR1 and LjCLE-RS1/2, acts
to inhibit auxin accumulation. Moreover, a simultaneous analysis
of the constitutive activation of LjCLE-RS genes in relation to
auxin accumulation and cortical cell division patterns shows that
signaling of these genes negatively regulates nodule development.
We suggest that this effect might occur as a result of the blocking
of further proliferation of cortical cells, probably by controlling
auxin accumulation, although initiation of cell division is unlikely
to be under the same control.
Development 139 (21)
Expression analysis
The primers used are listed in supplementary material Table S2. LjTAA1
(chr2.CM0008.610) and LjTAR1 (chr2.CM0008.590) were identified by a
BLAST search of the L. japonicus genomic sequence database using the
amino acid sequence of Arabidopsis TAA1 as the query. Total RNA was
isolated from each plant tissue using the RNeasy Plant Mini Kit (Qiagen).
First-strand cDNA was prepared using a QuantiTect Reverse Transcription
Kit (Qiagen). Real-time RT-PCR was performed using an ABI Prism 7000
(Applied Biosystems) with THUNDERBIRD SYBR qPCR Mix (Toyobo)
according to the manufacturer’s protocol. The expression of ubiquitin was
used as the reference (Takeda et al., 2009). Data show the mean (± s.d.) of
three biological replicates.
Microscopy
Bright-field and fluorescence microscopy were performed with an SZX12
stereomicroscope (Olympus) or with an A1 confocal laser-scanning
microscope (Nikon). Images were acquired and analyzed using DP
Controller (Olympus), NIS Elements (Nikon) or Photoshop (Adobe
Systems).
RESULTS
Auxin accumulates predominantly in dividing
cortical cells during nodule development
In order to determine the precise distribution of auxin during
nodule development in L. japonicus, we created stable transgenic
plants that express a GFP and nuclear localization signal (NLS)
fusion protein (GFP-NLS) under the control of DR5, which is a
highly active synthetic auxin-responsive element (Ulmasov et al.,
1997). Strong GFP expression was observed in the root apex
including the putative quiescent center, and during lateral root
development, as has been reported in other plants (supplementary
material Fig. S1A-C) (Benková et al., 2003). In addition, GFP
expression was induced by exogenous application of auxin
(supplementary material Fig. S1D). Our analysis showed that auxin
distribution patterns during nodule development could be indirectly
monitored in DR5::GFP-NLS transgenic plants.
The Mesorhizobium loti strain MAFF303099, which
constitutively expresses DsRED, was used to visualize rhizobia in
developing nodules following infection of plant roots. Three days
after inoculation (dai) with rhizobia, infection threads had started
to form in some root hairs, and GFP expression was observed in a
small number of cortical cells beneath root hairs with infection
threads (Fig. 1A,E). The nuclei in the cortical cells immediately
before cell division appeared larger than those of surrounding cells.
In roots at 5 dai, variable degrees of cortical cell proliferation had
occurred and this cell division was coincident with strong GFP
expression (Fig. 1B,C,F,G). Strong GFP expression continued until
the actively dividing cortical cells produced the initial bulge of the
nodule primordia, which was invaded by rhizobia via the growing
infection threads (Fig. 1D,H). After rhizobial colonization of the
developing nodule primordia, the strong GFP expression halted in
the infected region of the nodule and was restricted to the
surrounding tissues (Fig. 1I). This pattern was maintained after the
nodules enlarged and the rhizobia expanded their region of
infection (Fig. 1J). At this stage, GFP expression was also
observed at the vascular bundle (lenticels).
Next, we carried out an in situ hybridization analysis to confirm
the GFP expression patterns described above. This analysis showed
that GFP transcripts were detectable in proliferating cortical cells
(Fig. 1K); a similar expression pattern was found for the S-phasespecific marker HISTONE H4 (Fig. 1L). In developing nodules
with rhizobial colonies, GFP transcripts were distributed around
the region of infection (Fig. 1M). These distribution patterns were
RESEARCH ARTICLE 3999
consistent with the results of the GFP fluorescence analysis
described above.
It has been reported that the CYCLOPS gene is specifically
expressed in dividing cortical cells during nodulation in L.
japonicus (Yano et al., 2008). We performed hairy root
transformation of DR5::GFP-NLS transgenic plants in order to
obtain expression of mCherry-NLS under control of the CYCLOPS
promoter. A co-localization analysis of DR5::GFP-NLS and
pCYCLOPS::mCherry-NLS showed that GFP and mCherry are
expressed in the same cells during nodule development (Fig. 1NP). This suggests that auxin accumulation occurs in cells in which
the nodulation-related gene is expressed. We therefore conclude
that auxin accumulation during nodule development occurs
predominantly in proliferating cortical cells.
During nodule development, infection thread formation and
cortical cell proliferation are closely related (Murray, 2011). To
further investigate the relationship between infection thread
formation and auxin accumulation, we examined auxin distribution
patterns in a cyclops mutant (Yano et al., 2008). In contrast to wild
type (WT), in which the infection threads grew through root hairs
towards the cortical cells, they did not reach the cortical cells in the
cyclops-6 mutant (Fig. 2A,B). This failure of infection thread
growth is the likely cause of the premature arrest of nodule
development in the mutant (Fig. 2C,D). DR5::GFP-NLS/cyclops6 plants were produced by crossing DR5::GFP-NLS transgenic
plants with cyclops-6 plants. During nodulation in DR5::GFPNLS/cyclops-6 plants, however, induction of GFP expression was
observed in cortical cells below the root hairs where the defective
infection threads were located (Fig. 2F). GFP was still expressed
in the developmentally arrested nodules formed in the mutant (Fig.
2G). These results suggest that it is not necessary for the infection
threads to reach the cortical cells for the local accumulation of
auxin to occur.
Cytokinin signaling regulates auxin accumulation
during nodule development
In L. japonicus plants carrying the dominant snf2 mutation, the
constitutive activation of the cytokinin signaling pathway causes
spontaneous nodule formation in the absence of rhizobia (Fig. 2E)
(Tirichine et al., 2007). To clarify the relationship between auxin
accumulation and cytokinin signaling during nodule development,
auxin distribution patterns were analyzed in snf2 mutant plants.
DR5::GFP-NLS/snf2 plants were produced by crossing
DR5::GFP-NLS transgenic plants with snf2 plants. During
spontaneous nodule formation in DR5::GFP-NLS/snf2, induction
of GFP expression was observed in the cortical cells that were
proliferating to form the primordia of spontaneous nodules (Fig.
2H). GFP expression was maintained in the inner region of
growing spontaneous nodules even when they were almost as large
as normal nodules formed following rhizobial infection. In the
latter, GFP expression is excluded from the inner regions colonized
by the rhizobia (Fig. 1J; Fig. 2I). Thus, auxin accumulation patterns
during spontaneous nodule development in the snf2 mutant suggest
that cytokinin signaling positively regulates auxin accumulation
and that rhizobia might have a role in the exclusion of auxin from
infected cells.
NIN is involved in the local accumulation of auxin
NIN encodes an RWP-RK type transcription factor that acts in the
downstream part of the cytokinin signaling pathway (Schauser et
al., 1999; Tirichine et al., 2007; Madsen et al., 2010). To elucidate
the relationship between NIN and auxin, we analyzed auxin
DEVELOPMENT
Auxin involvement in nodulation
4000 RESEARCH ARTICLE
Development 139 (21)
distribution patterns in a nin mutant and in the roots of transgenic
plants that constitutively express NIN. In the nin-9 mutant,
nodulation was completely inhibited, although excessive curling of
root hairs was observed, as has been reported previously for other
nin alleles (Schauser et al., 1999). DR5::GFP-NLS/nin-9 plants
were produced by crossing DR5::GFP-NLS transgenic plants with
nin-9 plants. No specific distribution of GFP was observed in the
cortical cells beneath the curled root hairs in DR5::GFP-NLS/nin9 (Fig. 3A). When NIN was expressed under the control of the L.
japonicus ubiquitin promoter (pLjUBQ) in the hairy root of
DR5::GFP-NLS transgenic plants, ectopic division of cortical cells
was induced. The cortical cells formed nodule- and lateral root-like
organs in the absence of rhizobia (Fig. 3B,C; T. Soyano and M.
Hayashi, personal communication). During formation of these
structures, localized expression of GFP was observed in the cortex
of DR5::GFP-NLS roots that constitutively expressed NIN (Fig.
3D). This pattern of GFP expression continued in the bulge formed
by the nodule/lateral root-like organs (Fig. 3E,F). Thus, our
observations indicate that NIN plays a role in the local
accumulation of auxin in the cortical cells of roots and that this
probably leads to the activation of cortical cell division.
Autoregulation of nodulation controls auxin
accumulation
Autoregulation of nodulation (AON) is a presumptive systemic
regulatory mechanism that controls nodule number (Oka-Kira and
Kawaguchi, 2006). We investigated the interaction between auxin
and key components of AON, specifically, the HAR1 and LjCLE-
DEVELOPMENT
Fig. 1. Auxin accumulation patterns during wild-type nodule development. (A-J)Auxin accumulation patterns during nodule development
are indirectly shown by GFP expression (green) in DR5::GFP-NLS L. japonicus plants. Fluorescence and nodule formation were observed from front
(A-D) and side (E-J) views of infected roots. Red areas indicate the presence of rhizobia. (K-M)In situ localization of GFP (K,M) and HISTONE H4 (L)
in DR5::GFP-NLS plants. Arrow indicates the region of rhizobial colonization. (N,O)DR5::GFP-NLS plant that has transgenic hairy roots containing
the pCYCLOPS::mCherry-NLS construct. (P)A merged image of N and O. Yellow signals indicate co-localization of GFP (N, green) and mCherry (O,
red). Plants were inoculated with M. loti MAFF303099 that constitutively expressed DsRED (A-M) or with M. loti MAFF303099 (N-P). Roots were
observed 3 (A,E), 5 (B,C,F,G,K,L,N-P), 8 (D,H), 10 (I) and 12 (J) days after inoculation (dai) of rhizobia. For fluorescence analysis, at least 20 plants
(A-J) or 15 plants having transgenic hairy roots (N-P) were analyzed at each developmental stage in three independent experiments. Scale bars:
100m.
Fig. 2. Auxin accumulation patterns in cyclops and snf2 mutants.
(A,B)Infection thread formation in wild-type (WT) (A) and cyclops-6 (B)
L. japonicus plants at 7 dai. (C,D)Nodules in WT (C) and cyclops-6 (D)
at 14 dai. (E)Spontaneous nodule in snf2 35 days after germination
(dag) in the absence of rhizobia. (F,G)GFP expression patterns during
nodule formation in DR5::GFP-NLS/cyclops-6 at 7 (F) and 14 (G) dai.
(H,I)GFP expression patterns during spontaneous nodule formation in
DR5::GFP-NLS/snf2 at 14 (H) and 28 (I) dag in the absence of rhizobia.
Fluorescence, nodules and spontaneous nodule formation were
observed using side views of the root (F-I). Arrows indicate premature
growth arrest of infection threads. Arrowhead indicates premature
arrest of nodule formation. For fluorescence analysis, at least 20 plants
were analyzed at each developmental stage in three independent
experiments. Scale bars: 100m in A,B,F-I; 1 mm in C-E.
RS genes. Compared with the WT, the number of nodules increased
within a much larger nodulation zone in har1 mutants (Fig. 2C;
Fig. 4A). DR5::GFP-NLS/har1-8 plants were produced by crossing
DR5::GFP-NLS transgenic plants with har1-8 plants. In 5-dai roots
of DR5::GFP-NLS/har1-8, GFP was not only strongly expressed
but also expressed more widely than in DR5::GFP-NLS/WT plants
(Fig. 1C,G; Fig. 4B,C). The GFP expression pattern in the har1
mutant suggests that excessive induction of cortical cell division
leads to the increased number of nodules and that this is associated
with a considerable increase in auxin accumulation in the mutant.
Overall, our findings suggest that HAR1 might function to
negatively regulate the accumulation of auxin.
LjCLE-RS1 and LjCLE-RS2, which encode small CLE peptides,
are presumed to act as root-derived negative regulatory signals that
function via receptor complexes, including HAR1, during
nodulation (Okamoto et al., 2009; Kouchi et al., 2010). Although
the expression of LjCLE-RS1 and LjCLE-RS2 is significantly
increased in roots immediately after rhizobial infection, constitutive
expression of either gene can suppress nodule formation (Okamoto
et al., 2009). We therefore sought to identify the site of action of
the negative regulation resulting from activation of the LjCLE-RS
genes and to investigate its relationship to auxin accumulation. In
order to monitor cell division patterns, we made use of LjLTI6b
(the putative L. japonicus ortholog of Arabidopsis LTI6b) because
RESEARCH ARTICLE 4001
Fig. 3. Relationship between NIN expression and auxin
accumulation. (A)GFP expression patterns in DR5::GFP-NLS/nin-9 L.
japonicus plants at 7 dai. (B,C)Non-symbiotic root phenotype in
DR5::GFP-NLS plants that have transgenic hairy roots, in which NIN and
mKO2 are constitutively expressed. Transgenic roots were identified by
means of mKO2 expression (C). Arrowheads indicate nodule/lateral
root-like organs. (D-F)GFP expression patterns during development of
the nodule/lateral root-like organs formed in DR5::GFP-NLS plants that
have transgenic hairy roots constitutively expressing NIN in the absence
of rhizobia. Arrow indicates locally accumulated GFP signal in cortical
cells. Fluorescence was observed using side views of the root (A,D-F).
For fluorescence analysis, at least 15 plants (A) or 15 plants having
transgenic hairy roots (B-F) were analyzed at each developmental stage
in three independent experiments. Scale bars: 100m in A,D-F; 1 mm
in B,C.
LTI6b has been reported to be localized at the plasma membrane
(see Materials and methods) (Reddy et al., 2004). The plasma
membrane of host plants could be labeled by expressing a GFPLjLTI6b fusion protein under control of the 35S promoter
(Fig. 5A).
Nodulation phenotypes were examined in DR5::GFP-NLS plants
that carried either pLjUBQ::LjCLE-RS1 or LjCLE-RS2;
p35S::GFP-LjLTI6b in their hairy roots. In comparison to the hairy
roots of control plants that expressed the GUS gene, constitutive
expression of either of the LjCLE-RS genes reduced the number of
nodules (Fig. 4D-F; supplementary material Fig. S2A) (Okamoto
et al., 2009). Infection thread formation was observed in almost all
hairy roots of control plants (19/20; Fig. 4D; Fig. 5A). Importantly,
although nodule formation was suppressed by constitutive
expression of LjCLE-RS genes, infection thread formation was
observed in 85% of the plants (17/20; Fig. 4E; Fig. 5F), suggesting
that the rhizobial infection process was unaffected by activation of
the LjCLE-RS genes.
Comparison of cortical cell division patterns and auxin
distribution in control roots showed that auxin accumulation was
closely related to the progress of bulge formation in the nodule
primordia (Fig. 5A-C). In hairy roots of plants constitutively
expressing LjCLE-RS genes, auxin accumulation patterns
immediately before the initiation of cortical cell proliferation were
indistinguishable from those of control hairy roots (Fig. 5A,F).
Furthermore, we noticed that some initial cortical cell divisions
were accompanied by auxin accumulation in the roots (Fig. 5G;
supplementary material Fig. S2B). Initial cortical cell division was
observed in 21/25 pLjUBQ::LjCLE-RS1 plants and 17/20
DEVELOPMENT
Auxin involvement in nodulation
4002 RESEARCH ARTICLE
Development 139 (21)
Fig. 4. Auxin accumulation patterns in the har1 mutant and the effects of constitutive activation of LjCLE-RS genes on nodulation.
(A)Nodulation phenotype of har1-8 L. japonicus plants at 14 dai. (B,C)GFP expression patterns during nodule development of DR5::GFP-NLS/har18 plants at 5 dai. Fluorescence and nodule formation were observed using front (B) or side (C) views of infected roots. For fluorescence analysis, at
least 20 plants were analyzed at each developmental stage in three independent experiments. (D,E)Nodulation phenotype of DR5::GFP-NLS plants
that have transgenic hairy roots constitutively expressing both GUS and GFP-LjLTI6b (D), or both LjCLE-RS1 and GFP-LjLTI6b (E). Transgenic roots
were identified by their widespread distribution of GFP signals, derived from GFP-LjLTI6b, compared with those of GFP-NLS that are localized in
specific regions of the root. Infection threads appear as red spots in the roots. (F)Nodule numbers at 16 dai in DR5::GFP-NLS plants that have
transgenic hairy roots constitutively expressing LjCLE-RS genes (n13-17). Error bars indicate s.d. Scale bars: 1 mm in A,D,E; 100m in B,C.
Overall, these findings indicate that activation of LjCLE-RS genes
may act to inhibit further cortical cell divisions; the initial cortical
cell divisions appear to avoid this negative regulation.
The expression of a TAA-like gene is induced upon
rhizobial infection
The analyses described above identify the site of auxin
accumulation and place this in a genetic context with respect to the
regulation of nodule development. To determine the relationship
between auxin production and nodulation, we examined the
expression patterns of genes involved in auxin biosynthesis during
nodulation. We focused on the L. japonicus homologs of
TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)
and its paralog TRYPTOPHAN AMINOTRANSFERASE RELATED
Fig. 5. The effects of constitutive activation of LjCLE-RS1 on cortical cell division and auxin accumulation. (A-E)Infected DR5::GFP-NLS L.
japonicus plants that have transgenic hairy roots containing pLjUBQ::GUS and p35S::GFP-LjLTI6b. (F-I)Infected DR5::GFP-NLS plants that have
transgenic hairy roots containing pLjUBQ::LjCLE-RS1 and p35S::GFP-LjLTI6b. (J)Magnified image of I. Fluorescence and cortical cell division were
observed using front (E,G,I,J) or side (A-D,F,H) views of the roots. Plant plasma membrane was visualized by the expression of GFP-LjLTI6b. Green
dots indicate auxin accumulation. Arrows indicate the regions where vestiges of cell division with diminished auxin accumulation were observed.
Roots were at 6 (A,F) or 12 (B-E,G-J) dai. At least 20 plants having transgenic hairy roots were analyzed at each developmental stage in three
independent experiments. Scale bars: 100m.
DEVELOPMENT
pLjUBQ::LjCLE-RS2 plants. These results suggest that although
the numbers of nodules decreased, initial cortical cell division still
occurred in the presence of constitutive activation of LjCLE-RS
genes. However, we also frequently observed vestiges of cell
division with diminished auxin accumulation (Fig. 5H-J;
supplementary material Fig. S2C,D). Such vestiges were present in
22/25 pLjUBQ::LjCLE-RS1 plants and 18/21 pLjUBQ::LjCLE-RS2
plants. This implied that premature arrest of cortical cell divisions
occurred in hairy roots with constitutively activated LjCLE-RS
genes. In control hairy roots, vestiges of cell division were also
observed in 15/20 plants, although most cortical cell divisions
proceeded to form nodule primordia (Fig. 5C-E). Thus, arrest of
cortical cell divisions can also occur in WT nodule development in
which the AON mechanism is anticipated to be fully functional.
(TAR) because the TAA family produces indole-3-pyruvic acid
(IPA), which is converted to indole-3-acetic acid (IAA) by the
YUCCA family; this IPA pathway is thought to be the main route
for IAA biosynthesis (Mashiguchi et al., 2011; Won et al., 2011).
During nodulation, we found that expression of LjTAR1 was
transiently increased at 3 dai, which is when local auxin
accumulation started to occur, whereas LjTAA1 expression did not
change following rhizobial infection (Fig. 1A; Fig. 6A,B).
DISCUSSION
In this study, we focused on the patterns of auxin accumulation
during early nodule development, that is, the developmental stage
from the initiation of cortical cell proliferation to the formation of
nodule primordia. We show here that auxin predominantly
accumulates in dividing cortical cells. The observed auxin
distribution patterns are similar to those reported previously using
the GH3 promoter (Pacios-Bras et al., 2003; Takanashi et al.,
2011a); however, we have also been able to correlate auxin
accumulation patterns with various genetic factors that are key to
the formation of nodules.
Leguminous plants have two major and morphologically distinct
types of nodule: indeterminate and determinate (Ferguson et al.,
2010). In indeterminate nodules, such as those of Medicago and
Pisum, the nodule meristem can be distinguished at the tip of
nodules and this meristem stays active until the nodule becomes
senescent. By contrast, in determinate nodules, such as those in
Lotus, the activity of the nodule meristem appears to cease during
early nodule development, although the identity and location of the
nodule meristem is poorly characterized. Our observations apply
particularly to the development of determinate nodules. After
rhizobial colonization of developing nodules, the accumulation of
auxin becomes restricted to the region surrounding the infected
cells. This region appears to correspond to the nodule parenchyma,
which has previously been reported to show expression of organ
differentiation markers such as ENOD2 (van de Wiel et al., 1990;
Niwa et al., 2001). As the size of a determinate nodule continues
to increase by cell proliferation and elongation even after
differentiation, it is likely that the meristematic activities are
maintained within the nodule. Since the auxin distribution pattern
during nodule development in Lotus resembles that of meristematic
cells, the nodule parenchyma might be a candidate for the nodule
meristem. Further analysis, for example defining the cell division
Fig. 6. LjTAA-like gene expression patterns during nodulation.
Real-time RT-PCR analysis of (A) LjTAA1 and (B) LjTAR1 expression in
WT non-inoculated (0), 1, 3, 5 and 7 dai roots. Each cDNA was
prepared from total RNA derived from the whole root. Fold changes in
expression are shown relative to roots at 0 dai. Error bars indicate s.d.
RESEARCH ARTICLE 4003
patterns in the nodule, will be needed to clarify the identity and
location of the nodule meristem in determinate nodules. Auxin
accumulation occurs not only in dividing cortical cells but also in
the vascular bundle (lenticels) at later stages of nodule
development. The formation of lenticels is inhibited by auxin
transport inhibitors (Takanashi et al., 2011a; Takanashi et al.,
2011b).
During spontaneous nodule formation in the snf2 mutant, auxin
accumulation was observed at the inner nodular regions; by
contrast, these inner regions are occupied by rhizobia in normal
nodules. In comparison to normal nodules, some of the
spontaneous nodules formed in the snf2 mutant were distorted in
shape (data not shown). Thus, it is possible that persistent auxin
accumulation in the inner region causes excess cell division and
leads to the distorted shape of some spontaneous nodules.
Many of the genes that have been shown to be involved in the
positive regulation of nodulation function in the epidermis to signal
the occurrence of infection (Kouchi et al., 2010). However,
nodulation-related cytokinin receptors (LHK1 in L. japonicus and
CRE1 in M. truncatula) are specifically involved in nodule
development (Gonzalez-Rizzo et al., 2006; Murray et al., 2007;
Tirichine et al., 2007). The RWP-RK type transcription factor NIN
acts in the downstream part of the cytokinin signaling pathway
(Schauser et al., 1999; Tirichine et al., 2007; Madsen et al., 2010).
Recently, NIN has also been shown to be able to initiate cortical
cell division: constitutive activation of NIN induces ectopic
division of the cells in the absence of rhizobia (T. Soyano and M.
Hayashi, personal communication). Here, we investigated the
relationship between auxin accumulation patterns and the
expression of genes that encode positive regulators of nodule
development. Our results show that auxin accumulation was
induced when cytokinin signaling was constitutively activated.
Furthermore, in the roots of transgenic plants that constitutively
expressed NIN, local auxin accumulations were observed. Based
on these results, we propose a model for the molecular mechanism
that regulates cortical cell division through control of auxin
accumulation (Fig. 7). In the model, infection signals from the
epidermis ultimately activate cytokinin signaling in the cortex,
which causes a downstream transcription factor, NIN, to establish
local auxin accumulation in some cortical cells, which in turn
triggers division of these cortical cells. Since auxin accumulates in
the dividing cortical cells until the formation of nodule primordia,
then maintenance of auxin accumulation is required for nodule
organogenesis. The establishment of local auxin accumulation
appears to be related to the inhibition of polar auxin transport;
previous studies have shown that inhibition of polar auxin transport
in Medicago roots induces the formation of pseudonodules (Hirsch
et al., 1989; Rightmyer and Long, 2011) and that expression of
some auxin transporters is negatively regulated by cytokinin
signaling (Plet et al., 2011). Here, however, we found that
expression of a gene involved in auxin biosynthesis was
contemporaneous with the beginning of local auxin accumulation.
This observation suggests that inhibition of polar auxin transport
in conjunction with de novo auxin production might contribute to
the establishment of local auxin accumulation. Further
investigation to identify the interactions of genes involved in the
positive regulation of nodule development, polar auxin transport
and auxin biosynthesis would clarify the molecular mechanism
responsible for the local accumulation of auxin at the sites of
incipient nodule primordia.
In legumes, AON is known as a genetic mechanism that controls
the number of nodules via long-distance communication between
DEVELOPMENT
Auxin involvement in nodulation
Fig. 7. Model for the regulation of cortical cell division and the
site of auxin action in nodule development. The light-blue box
indicates the developmental stage of cortical cell division that is
negatively regulated by AON. The darker blue box beneath, which
partially overlaps the light-blue box, indicates the developmental stage
when maintenance of auxin accumulation occurs in all proliferating
cortical cells. Green and red regions of cortical cells indicate the
presumed sites of auxin accumulation and of rhizobia, respectively.
CCD, cortical cell division; SDI, shoot-derived inhibitor. See text for
explanation of the model.
the shoot and the root. Mutation or knockdown of the genes
involved in this feedback mechanism cause hypernodulation
phenotypes (Wopereis et al., 2000; Krusell et al., 2002; Nishimura
et al., 2002; Miyazawa et al., 2010; Mortier et al., 2012). In AON,
it has been proposed that root-generated signals are induced and
transported to the shoot upon infection by rhizobia. Two LjCLERS peptides have been identified as possible candidates for the
signals in L. japonicus (Okamoto et al., 2009). In M. truncatula,
cytokinin treatment induces the expression of MtCLE13, a
functional counterpart of the LjCLE-RS genes, and the effects are
abolished in nin mutants (Mortier et al., 2012). This suggests that
activation of AON-related CLE genes occurs downstream of NIN.
The root-derived signals are thought to be perceived by a receptor
complex that includes HAR1, which is a putative receptor-like
kinase, in the shoot; certainly, constitutive activation of LjCLE-RS
genes has no effect on nodulation in har1 mutant plants (Nishimura
et al., 2002; Okamoto et al., 2009). The output of LjCLE-HAR1
signaling, an unidentified shoot-derived inhibitor, might be
transported to the root to negatively regulate nodulation (Kouchi et
al., 2010; Reid et al., 2011b). Here, we showed that, compared with
the WT, the har1 mutant has increased and more widespread
accumulation of auxin in cortical cells. This indicates that auxin
accumulation prior to cortical cell division is controlled by the
AON mechanism (including HAR1) and that it determines not only
the auxin accumulation level but also the site of accumulation (Fig.
7; supplementary material Fig. S3).
Development 139 (21)
Although constitutive expression of AON-related CLE genes
in legumes causes inhibition of nodulation (Okamoto et al.,
2009; Mortier et al., 2010; Reid et al., 2011a), the site of the
negative regulation has remained elusive. Our simultaneous
observation of cortical cell division and auxin accumulation
patterns shows that early auxin accumulation and the initiation
of cell division can occur even in the presence of constitutively
activated LjCLE-RS genes. Thus, the developmental process does
not seem to be affected by constitutive expression of LjCLE-RS
genes. By contrast, we frequently observed vestiges of cortical
cell division, implying its premature arrest, accompanied by
reduced levels of auxin accumulation in transgenic roots.
Importantly, these cell division vestiges were also observed in
control roots, in which the LjCLE-RS genes were appropriately
activated by rhizobial infection. Thus, the premature arrest of
cortical cell division was not a secondary effect of constitutive
activation of LjCLE-RS genes, as it is probable that such arrest
occurs normally. Overall, we presume that activation of LjCLERS genes has a negative regulatory effect on cortical cell division
after its initiation, possibly through inhibiting the maintenance
of auxin accumulation (Fig. 7; supplementary material Fig. S3).
Further investigation needs to be carried out to determine
whether the link between activation of LjCLE-RS genes and
auxin accumulation is direct or indirect.
Both nodules and lateral roots are formed as lateral organs of
roots, and the regulatory mechanisms for these two organs seem to
have some components in common and others that are organ
specific (Desbrosses and Stougaard, 2011). On the basis of the
results obtained here, we propose the existence of a novel
molecular mechanism that controls cortical cell division in a
developmental stage-specific manner during nodule development.
This fine-tuning mechanism for regulation of cortical cell division
is reminiscent of lateral root development, in which organogenesis
initiation in the founder cells, the formation of primordia and lateral
root emergence are regulated by distinct factors involved in auxin
signaling (Benková and Bielach, 2010). In order to achieve a
greater understanding of the characteristic features of nodule
organogenesis, the identification of stage-dependent regulators of
cortical cell division is required.
Acknowledgements
We thank Jan Lohmann for the DR5 construct; Detlef Weigel for the GFP-NLS
and mCherry-NLS constructs; Makoto Hayashi for the pCYCLOPS construct and
for M. loti MAFF303099 expressing DsRED; Satoru Okamoto for LjCLE-RS1/2
constructs; Jens Stougaard for A. rhizogenes strain AR1193 and the nin-2 and
har1-3 mutants; and Takashi Soyano and M. Hayashi for sharing unpublished
data. Confocal images were acquired at the Spectrography and Bioimaging
Facility, NIBB Core Research Facilities.
Funding
This research was supported by Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan
[22870035 and 23012038 to T.S., 22128006 to M.K.], grants from The
NOVARTIS Foundation (Japan) for the Promotion of Science (to T.S.) and grants
from The Sumitomo Foundation (to T.S.).
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.084079/-/DC1
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