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Plant Signal Behav. 2010 April; 5(4): 403–405.
PMCID: PMC2958591

Analysis of two potential long-distance signaling molecules, LjCLE-RS1/2 and jasmonic acid, in a hypernodulating mutant too much love

Abstract

Legume plants tightly control the number and development of root nodules. This is partly regulated by a long-distance signaling known as autoregulation of nodulation (AON). AON signaling involves at least two potential long-distance signals: root-derived signal and shoot-derived signal. However, their molecular characteristics and the mode of action remain unclear. In our recent study, we isolated a novel Lotus japonicus hypernodulating mutant too much love (tml). Based on several grafting experiments, we concluded that its causative gene TML functions as a receptor of the shoot-derived signal. This finding prompted us to ask how the candidates of the long-distance signal molecules, LjCLE-RS1/2 and jasmonic acid (JA), are affected in tml mutants. Expression analysis revealed that rapid induction of LjCLE-RS1/2 upon rhizobial inoculation is still intact in tml, supporting that TML plays a role in reception of the shootderived signal but not in generation of the root-derived signal. Furthermore, physiological analysis showed that JA, a candidate of the shoot-derived signal, can suppress tml hypernodulation. Therefore, contrary to the previous report, JA might not be a component of AON signaling.

Key words: CLE, legume-Rhizobium symbiosis, long-distance signaling, Lotus japonicus, nodulation, HAR1

Leguminous plants develop root nodules to facilitate symbiotic interaction with nitrogen-fixing bacteria called rhizobia. Since excessive nodule formation can be deleterious to the plant growth, the number and development of nodules are tightly regulated by the host plants. One such a host-controlled mechanism is known as autoregulation of nodulation (AON). AON is a negative feedback system mediated by a long-distance signaling between shoots and roots. A current model of AON signaling has been proposed as follows.1,2 First, early nodulation events in roots induce unknown “root-derived signal”, which moves up to the shoots and activates the CLAVATA1-like receptor kinase (LjHAR1/MtSUNN/PsSYM29/GmNARK).36 In turn, another signal called “shoot-derived signal” is generated in the shoots and transported to the roots, resulting in suppression of further nodule formation.1,2 However, the validity of this model and the molecular characteristics of the proposed two long-distance signals remain largely unknown.

To better understand the molecular mechanism underlying AON, we recently isolated a novel hypernodulating mutant of Lotus japonicus named too much love (tml).7 The tml mutants develop an excessive number of nodules over the wide range of the roots.7 Our grafting experiments showed that this tml hypernodulation phenotype is determined by the root genotype, suggesting that its causative gene TML is likely to function in the roots.7 Furthermore, inverted-Y-shaped grafting (i.e., grafting between one shoot and two roots) revealed that the effect of TML on nodulation is local but not systemic.7 Together, we proposed a model in which a root regulator TML may play a role as a receptor of the AON-associated shoot-derived signal.7

If indeed TML is such a receptor of the shoot-derived signal as we proposed, the tml mutation should not abolish induction of the root-derived signals. Several lines of evidence suggest that two L. japonicus CLE genes, LjCLE-Root Signal (RS) 1 and LjCLE-RS2, might encode such a root-derived signal.8 First, LjCLE-RS1/2 transcripts are rapidly upregulated in roots within 24 hr of rhizobial inoculation.8 Second, overexpression of LjCLE-RS1/2 in wild-type roots strongly suppresses nodulation.8 Finally, this inhibitory effect cannot be observed in har1 mutants.8 Given that HAR1 is the homolog of Arabidopsis CLV1 which recognizes a CLE peptide processed from CLAVATA3,3,4,9 it can be speculated that LjCLE-RS1/2-derived CLE peptides might potentially move up to shoots and directly activate HAR1.

Thus, we examined LjCLE-RS1/2 expression in tml mutants. Wild-type and tml plants were grown for 4 days and inoculated with Mesorhizobium loti MAFF303099. Like wild type, rapid induction of LjCLE-RS1 and LjCLE-RS2 was observed in tml roots at 24 hrs post inoculation (hpi) (Fig. 1A and B). Therefore, TML is not likely to participate in LjCLE-RS1/2 induction itself, further supporting the model that TML function as a receptor of the shoot-derived signals rather than a generator of the root-derived signals. Interestingly, the expression levels of LjCLE-RS1/2 at 24 hpi in tml mutants were much higher than those in wild type (Fig. 1A and B), suggesting that there might be a negative feedback regulation in the LjCLE-RS1/2 transcription.

Figure 1
LjCLE-RS1/2 expression in wild type and tml. Wild-type MG-20 and tml plants were grown in vermiculite for 4 days and inoculated with Mesorhizobium loti MAFF303099. Expression levels of LjCLE-RS1 (A) and LjCLE-RS2 (B) in the roots at 0 and 24 hr post inoculation ...

Finally, we analyzed the effect of jasmonic acid (JA) on nodulation in tml mutants. JA is a well-known plant hormone that regulates many aspects of plant immunity and development.10 It has been shown that exogenous application of JA leads a drastic decrease in nodule number in L. japonicus11 as well as Medicago truncatula.12 More importantly, this inhibitory effect of JA on nodulation was also observed in har1 hypernodulating mutants; hence, JA has been proposed as a potential shoot-derived signal that might act downstream of HAR1.11 If this model is correct, JA should not affect tml hypernodulation, which is presumably due to a defect in the shoot-derived signal perception.

To test this hypothesis, we treated aerial parts of wild type and tml with JA at different concentrations and counted nodules and nodule primordia at 12 days post inoculation. As Figure 2 shows, the number of nodules and nodule primordia was reduced in tml as well as wild type in a dose-dependent manner and 10−3 M of JA almost completely suppressed nodule development in both plants. This observation suggests that JA is not a signal molecule that mediates the shoot-to-root communication in the HAR1/TML pathway, disproving the previously-proposed model. Rather, JA is likely to act in an unknown pathway parallel to the HAR1/TML signaling pathway. It is also noted that as high as 10−3 M of JA is required for complete inhibition of nodulation at least in L. japonicus (Fig. 2). Considering that endogenous JA level is supposed to be much lower than 10−3 M in living plants, the inhibitory effect of this high concentration of JA could be an artifact.

Figure 2
Effect of JA on tml hypernodulation. Methyl jasmonic acid (MeJA) at different concentrations (0~10−3 M) was sprayed to aerial parts of wild type and tml 1 day prior to, and 1 and 6 day post Mesorhizobium loti MAFF303099 inoculation. Nodulation ...

In conclusion, the tml mutation did not affect induction of LjCLE-RS1/2, the strong candidates of the AON-associated root-derived signal molecules. This is consistent with our previous model that TML functions in reception of the shoot-derived signal but not in generation of the root-derived signal. Moreover, exogenous JA application can suppress not only har1 hypernodulation but also tml hypernodulation, indicating that JA is unrelated to the HAR1/TML pathway. Further investigation will be needed to elucidate the bona fide shoot-derived signal and the detailed molecular function of LjCLE-RS1/2 gene products.

Acknowledgements

This work was supported by Grant-in-Aid for Science Research for Priority Areas (18056004) and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan. S.M. was funded in part by Grant-in-Aid for JSPS Fellows.

Abbreviations

AON
autoregulation of nodulation
CLE
clavata3/ESR-related
CLV1
clavata1
HAR1
hypernodulation aberrant root formation 1
JA
jasmonic acid
LjCLE-RS1/2
Lotus japonicus CLE-root signal 1/2
TML
too much love

Footnotes

References

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