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Chapter
44
Lotus japonicus Nodulates When It
Sees Red
Maki Nagata
Department of Agricultural Sciences, Faculty of Agriculture, Saga University, Saga, Japan
Ann M. Hirsch
Department of Molecular, Cell and Developmental Biology and Molecular Biology
Institute, University of California, Los Angeles, CA, USA
Akihiro Suzuki
Department of Agricultural Sciences, Faculty of Agriculture, Saga University, Saga, Japan;
United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima,
Japan
44.1
INTRODUCTION
In nature, many legumes develop nodules on their roots in
which rhizobia fix atmospheric nitrogen into ammonia by
means of the enzyme nitrogenase. In return, the rhizobia
obtain photoassimilates from the host that had been fixed
within plant leaves, demonstrating that light quantity is critical for the effectiveness of this symbiosis. However, light
quality also has a tremendous influence on the success of the
legume–rhizobial symbiosis. In this chapter, we review the
literature on the effects of light quality on the establishment
of the legume–rhizobial symbiosis and describe our own
work to update our current knowledge concerning nodule
development and light.
44.2
RESULTS AND DISCUSSION
Numerous reports exist in the literature about sufficient light
quantity being critical for effective root nodule formation
(Fred and Wilson, 1934; Fred et al., 1938). In addition,
adding glucose and sucrose to the nutrient medium when
light was limiting enhanced root nodule formation in some
legumes (Van Schreven, 1959). These observations support
the idea that the accumulation of photoassimilates provides
energy and compounds important not only for root nodule formation but also for nodule function to maintain a
successful mutualism between the two symbiotic partners.
If we consider the fact that chlorophyll absorbs both red
(R; 600–700 nm) and blue light (400–500 nm), we can conclude that the difference in light quality (wavelength) also
affects the photosynthetic activity leading to the establishment of the symbiosis. The fact that the effects of light quality
on symbiosis are independent of photosynthetic activity has
been reported by a number of investigators. Lie (1964) studied the effects of short-time irradiation of R or far-red (FR)
light at the end of the photoperiod of the light cycle of the
day (end-of-day; EOD) on pea and kidney bean root nodule
formation, and reported that root nodule formation following irradiation with FR light was suppressed compared to
R light treatment (Lie, 1964). This phenomenon – that root
nodule formation is suppressed by irradiating FR light at the
EOD – was reported not only for pea (Lie, 1969) but also for
broad beans (Lie, 1971), soybean (Kasperbauer et al., 1984;
Hunt et al., 1987), and southern pea (cowpea) (Kasperbauer
and Hunt, 1994). In contrast, Balatti and Montaldi (1986)
showed that the number and biomass of soybean root nodules
Biological Nitrogen Fixation, Volume 1, First Edition. Edited by Frans J. de Bruijn.
© 2015 John Wiley & Sons, Inc. Published 2015 by John Wiley & Sons, Inc.
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Chapter 44
Lotus japonicus Nodulates When It Sees Red
20 days after inoculation with Bradyrhizobium japonicum
were significantly increased following FR irradiation in EOD
compared to R irradiation. The reasons for these contradictory results are unclear, but may depend on light quantity or
the exact timing of the irradiation. Nevertheless, root nodule formation was restored by the irradiation of R after FR
treatment in EOD (FR + R) and repressed once again following a light treatment of FR + R + FR in EOD. Because these
results illustrate the classic photoreversibility of FR with R,
the involvement of phytochrome in root nodule formation
was strongly indicated (Lie, 1964; Hunt et al., 1987; Kasperbauer and Hunt, 1994).
That phytochrome participates in the perception of
the R/FR ratio and the shade avoidance syndrome (SAS)
is well known. Plants have photoreceptors that sense the
presence of their neighbors by monitoring the ratio of R,
mainly absorbed by chlorophyll, to FR light, which is not.
A low R/FR ratio indicates the presence of neighbors that
may be competitors for photosynthetically active radiation
(PAR), thereby initiating the SAS, such that plant crowding
causes plants to grow taller or to bend to the light to avoid
being shaded (Smith and Whitelam, 1997; Neff et al., 2000;
Franklin, 2008; Franklin and Quail, 2010). However, these
low R/FR light conditions are suboptimal for root nodule
formation. Kasperbauer and Hunt (1994) investigated the
effect of the difference of R/FR ratio on southern pea root
nodule formation by covering the surface of the pots with
either gray-white or brick-red soil. Because the spectral
distribution of light reflected from different soils is not the
same, the R/FR ratio radiated to each pot through its covering differed. The final results showed that the number of root
nodules 18 days after inoculation was higher when plants
were grown under high R/FR (gray-white soil) than under
low R/FR (brick-red) conditions (Kasperbauer and Hunt,
1994). In another experiment, this time in the field where
multiple plants were planted together, the R/FR light ratios
that these plants perceived depended not only on the degree
of shading but also on row orientation because chlorophyll
absorbs R light more efficiently than FR light. Root nodule
formation for soybean and southern peas planted in either a
north–south (low R/FR) or east–west (high R/FR) orientation
was investigated. For both species, plants positioned in an
east–west row orientation produced more nodules than those
orientated north–south. In other words, plants grown under
high R/FR ratios produced more nodules (Hunt et al., 1990;
Kasperbauer and Hunt, 1994). However, the photosynthetic
photon flux density (PPFD) for plants exposed to high R/FR
conditions was higher than that for low R/FR exposure in
both sets of experiments. Therefore, these investigators did
not eliminate the possibility that root nodule number was
affected by photosynthetic activity and the greater allocation
of photoassimilates. Based on these results, we reasoned that
root nodule formation was linked to the perception of the
R/FR ratio and used Lotus japonicus (Suzuki et al., 2011;
Shigeyama et al., 2012) to test this hypothesis.
To verify that root nodule formation is under the control
of the perception of the R/FR ratio, we examined nodule
formation in L. japonicus MG20 wild-type and phyB mutant
plants grown under different R/FR light conditions. In
our study, the PPFD of the R light-emitting diode (LED)
remained constant to eliminate differences in the amount
of photosynthate produced under low R/FR and high R/FR
treatments. Signaling via phyB, the major red light photoreceptor used during seedling development, regulates
responses to photoperiodism, end-of-day FR, and R/FR
ratios in white light-grown plants (Neff et al., 2000; Franklin
et al., 2003). From our experiments, we determined that the
shoot and root fresh weights of phyB mutants were significantly reduced compared to those of MG20 plants grown
under white light conditions. In addition, the number of root
nodules of the phyB mutants was lowered compared to that
of MG20 plants. To better understand the effect of the R/FR
ratio on plant growth, we measured the fresh weights of the
aerial plant parts, root length, and root nodule number under
different R/FR light conditions. We found no statistically
significant difference for either root length or shoot fresh
weight for L. japonicus phyB mutants or MG20 plants,
respectively, when the plants were grown under high R/FR
and low R/FR light conditions. However, very few nodules
developed on the phyB mutant roots under different R/FR
light conditions. In addition, although nodules developed on
MG20 plants grown in high R/FR light, very few nodules
were detected on the roots of the low R/FR-grown plants.
Sucrose, a highly soluble disaccharide that provides
energy sources for plant cells (Huber, 1989), is synthesized
from the primary products of photosynthesis. To investigate
whether a difference in photosynthetic activity was present
while PPFD is constant, the sucrose content of the roots was
measured after the plants were grown under different R/FR
light conditions. We found that the sucrose content of roots
in low R/FR-grown plants increased compared to the high
R/FR-grown plants, suggesting FR light irradiation may be
responsible for the increase in sucrose content. Nevertheless,
the fact that root nodule number was reduced in the low
R/FR-grown plants in spite of the higher sucrose content
strongly suggests that phyB sensing of the R/FR ratio has
a significant influence on nodulation. However, we cannot
completely eliminate the possibility that photosynthetic
products contribute to root nodule formation when the plants
are grown under different R/FR light conditions.
Because the number of root nodules for both low
R/FR-grown MG20 plants and white light-grown phyB
mutants decreased, we hypothesized that phyB-mediated
signaling in plants controlled by root nodule formation.
Robson et al. (2010) suggested that phyA and JA signaling
cooperate to regulate the balance between shade avoidance responses in FR-enriched light and defense responses
44.2 Results and Discussion
to mechanical damage or herbivores. In low R/FR light,
phyB signaling suppressed both JA-mediated gene expression and JA-dependent defenses against insect herbivory
(Moreno et al., 2009). By contrast, other reports indicate
that antagonistic interactions between JA-mediated defense
signaling and chromophore-mediated light signaling exist.
For example, mutations in either HY1 or HY2, which encode
a phytochromobilin synthase, enhanced JA production and
sensitivity (Zhai et al., 2007). For root nodule formation, JA
has been reported to be a negative regulator. In L. japonicus,
shoot-applied methyl jasmonate (MeJA) strongly suppressed
the early stages of nodulation, including infection thread
formation and NIN expression, and also inhibited lateral root
formation (Nakagawa and Kawaguchi, 2006). In Medicago
truncatula, JA also suppressed root nodule development,
at an even lower concentration (Sun et al., 2006). Thus,
we predicted that JA production and/or sensitivity would
be enhanced in low R/FR-grown MG20 plants and white
light-grown phyB mutants. We analyzed the expression
of JA-responsive genes (PDF1.2, JAR1, and MYC2) in L.
japonicus grown under different R/FR light conditions. The
expression of PDF1.2, JAR1, and MYC2 genes decreased
in low R/FR-grown MG20 plants compared with plants
grown under high R/FR light conditions. Moreover, their
expression levels declined in phyB mutants under white light
conditions in contrast to MG20 plants. These results strongly
suggested that JA production and/or sensitivity decreased in
low R/FR-grown MG20 plants and white light-grown phyB
mutants, which was contrary to our expectations. To check
whether JA concentration correlated with the expression of
JA-responsive genes, we measured the endogenous concentrations of JA and JA-Ile in roots grown under white light
condition. We found no significant difference in endogenous
JA concentration between MG20 wild-type plants and phyB
mutant plants. However, the endogenous concentration of
JA-Ile significantly decreased in roots of phyB mutants.
JA-Ile, the isoleucine conjugate of JA, is a biologically
active hormonal signal (Staswick and Tiryaki, 2004) and is
generated by a JAR1-encoding JA-amino acid synthetase.
The JAR1 gene product is involved in pathogen defense,
sensitivity to ozone, and wound responses (Guranowski
et al., 2007; Staswick, 2008; Koo et al., 2009). Our results
might be explained by the fact that the conversion of JA to
JA-Ile was suppressed due to decreased JAR1 expression
in the phyB mutants grown under white light conditions,
which would give rise to a reduced concentration of JA-Ile
in the phyB mutants. Thus, we concluded that root nodule
formation was suppressed because the JA-Ile concentration
was low in plants grown under low R/FR light conditions.
To examine the effect of JA directly on root nodule formation under different R/FR light conditions, JA was added
to the plant growth medium. Both shoot and root growth
of MG20 plants were decreased as the concentration of JA
increased. Although the root nodule number per plant was
443
reduced after adding a high concentration (10 μM) of JA,
nodule number per plant for plants grown in low (0.1 μM)
JA significantly increased when compared to control plants
that received no exogenous JA. Furthermore, nodule number
per root length increased for plants treated with 0.1, 1, and
10 μM JA over the untreated controls. Our results differed
from those reported for M. truncatula (Sun et al., 2006), in
that in our study, we found that L. japonicus nodule number
was increased at low concentrations of JA.
If reduced nodule formation in low R/FR-grown MG20
plants and white light-grown phyB mutants is due to a
low concentration of endogenous JA-Ile, we predicted that
nodule formation would be enhanced by JA application. To
test this possibility, the effect of JA treatment on root nodule
development was analyzed. We found that the number of
root nodules and nodule primordia in 0.1 μM JA-treated
plants was slightly increased compared to untreated plants.
We also examined the expression of JA-responsive genes
and the NIN gene in 0.1 μM JA-treated or untreated plants.
NIN is required for infection thread formation and nodule
primordium initiation in L. japonicus (Schauser et al., 1999).
The expression of NIN gene was significantly increased
in 0.1 μM JA-treated plants. Although JA addition did not
affect the total root length, the number of infection threads
per root length was significantly increased in MG20 plants
compared to the untreated plants. These results strongly
suggest that low concentrations of JA function as a positive
regulator for root nodule formation in L. japonicus.
In addition, we analyzed the effect of JA treatment on
root nodule formation in phyB mutants. Following 0.1 μM
JA treatment of phyB mutants, shoot length was unaffected
and root length was decreased compared to the untreated
plants. However, the number of root nodules per plant significantly increased compared to the number in the untreated
plants. Moreover, nodule number per root length significantly increased in response to 0.1 and 1 μM JA treatments.
Based on the responses of JA treatments to shoot length,
total root length and root nodule number, the sensitivity of
MG20 plants and phyB mutants to JA is not likely to be
significantly different. To summarize, the cause of reduced
root nodule formation in low R/FR-grown MG20 plants and
white light-grown phyB mutants is due to the inhibition of
JA-Ile production.
The literature also reports about the effects of R/FR
ratios on the interaction between plants and microbes. For
example, low R/FR ratio reduced Arabidopsis resistance
to Botrytis cinerea (Cerrudo et al., 2012),‘ and fluorescent
illumination with a high R/FR ratio improved the resistance
of cucumber seedlings to powdery mildew (Sphaerotheca
cucurbitae) (Shibuya et al., 2011). Moreover, in the case of
the interaction between plants and pests, wild tobacco and
Arabidopsis grown under high R/FR conditions were less
susceptible to feeding damage by insect herbivory compared
to low R/FR-grown plants (Izaguirre et al., 2006; Moreno
444
Chapter 44
Leguminous plants grown
under the sunlight
High R/FR ratio
Phytochrome B
Lotus japonicus Nodulates When It Sees Red
Leguminous plants grown
under the shade
of another plants
Low R/FR ratio
Phytochrome B
JA signaling
SAS
JA signaling
Figure 44.1 Model representing the proposed
Root nodule
formation
SAS Root nodule
formation
Enhanced nodulation
Suppressed nodulation
et al., 2009). JA signaling is involved in these plant defense
mechanisms. Plants grown under high R/FR light produced
more JA and protected themselves from pathogens, whereas
plants grown under low R/FR light were more susceptible to
infection or feeding damage by pathogens and predators due
to the reduced production of or decreased sensitivity to JA
(Cerrudo et al., 2012; Moreno et al., 2009; Ballaré, 2011).
In root nodule symbiosis, the rhizobial infection process is
enhanced by this mechanism.
For plants, photosynthesis is the most important biochemical reaction for survival. Plants grow taller or bend
to light, but at the same time they assume the risk that they
are more likely to be invaded by pathogens. In low R/FR
light, phyB is inactivated by photoconverting the active
Pfr form into Pr, and as a result, phytochrome-interacting
factor 4 (PIF4) and 5 (PIF5) accumulate, leading to the
synthesis of auxin (IAA) and bioactive gibberellins (Ballaré,
2011), phytohormones required for plant growth. However,
the synthesis of phytohormones is incompatible with the
production of JA, making it very difficult for plants to
maintain active growth and high-level defense responses at
the same time. Legumes show the same energy constraints
by limiting the initiation of energy-costly nodules under low
R/FR conditions. However, by obtaining sufficient nitrogen
through the establishment of a symbiosis with rhizobia for
maintaining effective growth, legumes may ultimately be
better able to balance growth and defense than nonlegumes.
A model representing the proposed mechanism of JA
and phyB signaling for shade perception and root nodule formation is depicted in Figure 44.1.
In high R/FR light conditions, phyB suppresses SAS
and enhances root nodule formation through an increased
concentration of JA-Ile. In contrast, in low R/FR light conditions, SAS is restored by the inactivation of phyB and root
mechanism of JA and phyB signaling for shade
perception and root nodule formation. In high R/FR
light, phyB suppresses SAS and enhances root nodule
formation through an increased concentration of JA-Ile.
In low R/FR light, SAS is restored by the inactivation of
phyB, and root nodule formation is suppressed due to a
reduced concentration of JA-Ile. Small letters and dotted
lines mean inactivation and suppression, respectively.
nodule formation is suppressed due to the reduced concentration of JA-Ile. Our data show that root nodule formation
involves the perception of the R/FR light ratio and requires
signaling through phyB and JA. Although root nodule
development is initiated in the soil under low light, the R/FR
light conditions in soil are suboptimal for sustaining root
nodule function. Thus, host legumes shaded by other plants
initiate a shade avoidance response and modify their growth
to obtain sufficient light for maximizing photosynthesis.
Nevertheless, under such low R/FR light conditions, the host
plants suppress root nodule development to conserve energy.
We conclude that this SAS for root nodule formation is
required for L. japonicus nodule development. In conclusion,
sensing both light quality and quantity is essential for establishing and maintaining a successful nitrogen-fixation
symbiosis. How common this interaction is among the
symbiotic Fabaceae is not known at this time.
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