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Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus Satoru Okamoto

ab

& Masayoshi Kawaguchi

c

a

Division of Biological Science; Graduate School of Science; Nagoya University; Nagoya, Japan b

Research Fellow of the Japan Society for the Promotion of Science; Tokyo, Japan

c

Division of Symbiotic Systems; National Institute for Basic Biology; Okazaki, Japan Published online: 03 Jun 2015.

Click for updates To cite this article: Satoru Okamoto & Masayoshi Kawaguchi (2015) Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus, Plant Signaling & Behavior, 10:5, e1000138, DOI: 10.1080/15592324.2014.1000138 To link to this article: http://dx.doi.org/10.1080/15592324.2014.1000138

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SHORT COMMUNICATION Plant Signaling & Behavior 10:5, e1000138; May 2015; © 2015 Taylor & Francis Group, LLC

Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus Satoru Okamoto1,2,* and Masayoshi Kawaguchi3 1

Division of Biological Science; Graduate School of Science; Nagoya University; Nagoya, Japan; 2Research Fellow of the Japan Society for the Promotion of Science; Tokyo, Japan; 3 Division of Symbiotic Systems; National Institute for Basic Biology; Okazaki, Japan

Keywords: grafting, HAR1, long-distance signaling, nitrate, nodule

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Abbreviations: CLV1, CLAVATA1; CLE, CLAVATA3/ESR-related; HAR1, HYPERNODULATION ABERRANT ROOT FORMATION1; LRR-RK, Leucine-rich repeat receptor kinase; NARK, NODULE AUTOREGULATION RECEPTOR KINASE; SUNN, SUPER NUMERIC NODULES.

Nitrate is a major environmental factor in the inhibition of nodulation. In a model legume Lotus japonicus, a CLV1-like receptor kinase, HAR1, mediates nitrate inhibition and autoregulation of nodulation. Autoregulation of nodulation involves root-to-shoot-to-root long-distance communication, and HAR1 functions in shoots. However, it remains elusive where HAR1 functions in the nitrate inhibition of nodulation. We performed grafting experiments with the har1 mutant under various nitrate conditions, and found that shoot HAR1 is critical for the inhibition of nodulation at 10 mM nitrate. Combined with our recent finding that the nitrate-induced CLE-RS2 glycopeptide binds directly to the HAR1 receptor, this result suggests that CLE-RS2/HAR1 long-distance signaling plays an important role in the both nitrate inhibition and the autoregulation of nodulation.

Symbiotic nitrogen fixation in nodules is beneficial for host plants. However excessive nodulation inhibits plant growth because of the high energy cost of nitrogen fixation. Therefore, host plants control nodulation flexibly in response to environmental and internal stimuli. Nitrate is the most important environmental factor regulating nodulation.1 Lotus. japonicus HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1), Glycine max NODULE AUTOREGULATION RECEPTOR KINASE (NARK) and Medicago truncatula SUPER NUMERIC NODULES (SUNN) are involved in nitrate inhibition of nodulation,2–5 and encode leucine-rich repeat receptor kinase (LRR-RK) that show the highest similarity with CLAVATA1 (CLV1) among all Arabidopsis LRR-RKs.6–10 Mutants of these legume genes exhibit nitrate tolerant and hypernodulation phenotypes without altering the development of the shoot apical meristem. HAR1, NARK and SUNN are key factors in the rootto-shoot-to-root negative feedback regulation of nodulation, the so-called autoregulation of nodulation, and function in shoots to perceive a root-to-shoot long-distance signal.4,6–9 In nitrate inhibition of nodulation, SUNN is involved in the systemic control of nodulation, but whether shoots mediate this inhibition has remained unexplored.3 In soybean, there are studies suggesting that NARK mediates nitrate inhibition of nodulation in shoots,11,12 whereas Reid et al. (2011) highlighted the requirement for root NARK in nitrate inhibition of nodulation. Furthermore, local effects of nitrate on the symbiosis have been reported.13,14 These facts indicate that the mechanism behind

the nitrate response of nodulation might be complex. Recently, we showed that the L. japonicus CLE-RS2 glycopeptide can translocate from roots to shoots, and directly binds to the HAR1 receptor.15 Interestingly, the CLE-RS2 peptide gene responds not only to rhizobial inoculation but also to nitrate.16 This suggests that CLE-RS2 functions in both autoregulation and nitrate inhibition of nodulation. However, it is unknown whether shoot-root communication is essential for nitrate inhibition of nodulation in L. japonicus. To clarify this point, we performed grafting experiments in the presence of low and high levels of nitrate. L. japonicus WT and har1–4 hypernodulating mutant plants were used for grafting experiments. We confirmed that grafted plants consisting of shoot scions and rootstocks of the same genetic identity (WT/WT and har1/har1) showed similar phenotypes as the corresponding intact plant line.8,9 Then, we grafted har1 mutant shoot scions onto WT rootstocks (har1/WT) and WT shoot scions onto har1 mutant rootstocks (WT/har1). At low nitrate (0.5 mM KNO3), har1/WT grafted plants showed a hypernodulation phenotype, and WT/har1 grafted plants showed the normal nodulation phenotype as reported before8,9 (Fig. 1A). Interestingly, at high nitrate (10 mM KNO3), the number of nodules on har1/WT grafted plants was not decreased compared with har1/WT grafted plants at low nitrate (Fig. 1A and C). In addition, har1/WT grafted plants showed similar numbers of nodules as har1/har1 grafted plants even at 10 mM KNO3. This demonstrated that har1/WT plants are defective in nitrate

*Correspondence to: Satoru Okamoto; Email: [email protected] Submitted: 11/24/2014; Accepted: 12/09/2014 http://dx.doi.org/10.1080/15592324.2014.1000138

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Figure 2. Model of long-distance signaling in nitrate inhibition and autoregulation of nodulation. CLE-RS2 glycopeptide is induced by nitrate and rhizobial inoculation. CLE-RS2 glycopeptide is translocated to the shoot where it binds to the HAR1 receptor. Then, the shoot-derived inhibitor is sent to the roots.

Figure 1. Grafting experiment with L. japonicus B-129 (Gifu) and har1–4. The shoot-root grafting was performed as described.20,21 Five days after surgery, plants were transferred to vermiculite with 0.5 mM or 10 mM nitrate. The plants were placed under a 16-h light/8-h dark cycle at 24 C. Seven days after the transfer, the grafted plants were inoculated with Mesorhizobium loti. Fourteen days after the inoculation, the numbers of nodules were counted. (A) Number of nodules per plant in the presence of 0.5 mM and 10 mM KNO3. Error bars represent SD (n D 12 to 16). Statistical differences were evaluated using Student’s t-test. Asterisks indicate statistically significant differences (P < 0.01) between grafted plants grown in 0.5 mM and 10 mM KNO3. (B) Nodulation phenotype of a har1–4 root grafted to a wild-type shoot. (C) Nodulation phenotype of a wild-type root grafted to a har1–4 shoot. Roots in (B) and (C) were grown at 10 mM KNO3.

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inhibition of nodulation. By contrast, on WT/har1 grafted plants, the nodulation was strongly suppressed at 10 mM KNO3 (Fig. 1A and B). WT/har1 grafted plants showed similar inhibition of nodule number as WT/WT grafted plants. Evidently, the normal nitrate responsiveness had remained unaffected in WT/ har1 grafted plants. Thus, our grafting experiments revealed that shoot HAR1 plays a critical role in nitrate inhibition of nodulation. In addition, HAR1-independent morphological effects of nitrate were observed in nodules. Under 10 mM nitrate, all combinations of grafted plants formed small, white nodules (Fig. 1B and C), whereas, under low nitrate condition, nodules were pink (data not shown). These observations are consistent with those of Jeudy et al. (2010) and suggest that nitrate affects nodulation via both HAR1-dependent and -independent pathways. In this study, we showed that HAR1 in shoots mediates nitrate inhibition of nodulation. This result conforms to the finding that the HAR1 orthologous LRR-RK in M. truncatula, SUNN, mediates systemic nitrate inhibition of nodulation.3 Previously, we demonstrated that L. japonicus CLE-RS2 is responsive to both rhizobial inoculation and nitrate, and that overexpression of CLE-RS2 induces systemic suppression of nodulation in a HAR1-dependent manner.16 We also found that the CLE-RS2 glycopeptide directly binds to the HAR1 receptor and can be translocated from roots to shoots.15 Based on these findings, we propose a model for the long-distance nitrate inhibition of nodulation (Fig. 2). In this model, the CLE-RS2/HAR1 long-distance

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signaling system is shared between nitrate inhibition and autoregulation of nodulation. Reid et al.17 reported that root NARK mediates nitrate inhibition of nodulation. In soybean, a nitrate responsive CLE gene, NIC1, has been identified that, when overexpressed, suppresses nodulation locally in a NARK-dependent manner. Considering that the inhibitory effects of CLE-RS2 and NIC1 differ, the mode of action of nitrate responsive CLE peptides might affect the mechanism underlying the nitrate inhibition of nodulation. Nitrate is one of the essential nutrients for plants. Recently, it was reported that CLE peptides and CLV1 receptor are involved in nitrate response of root systems in Arabidopsis.18,19 CLV1 shows the highest similarity to HAR1 among the L. japonicus LRR-RKs.10 Considering that homologous molecules are involved in nitrate response in different families, there might be an evolutionary link between nitrate inhibition of nodulation References 1. Nutman PS. The Influence of the Legume in RootNodule Symbiosis. Biol Rev 1956; 31:109-51; http:// dx.doi.org/10.1111/j.1469-185X.1956.tb00650.x 2. Wopereis J, Pajuelo E, Dazzo FB, Jiang Q, Gresshoff PM, De Bruijn FJ, Stougaard J, Szczyglowski K. Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype. Plant J 2000; 23:97-114; PMID:10929105; http://dx.doi.org/10.1046/j.1365313x.2000.00799.x 3. Jeudy C, Ruffel S, Freixes S, Tillard P, Santoni AL, Morel S, Journet EP, Duc G, Gojon A, Lepetit M, et al. Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses. N Phytol 2010; 185:817-28; PMID:20015066; http://dx.doi.org/10.1111/j.14698137.2009.03103.x 4. Carroll BJ, McNeil DL, Gresshoff PM. A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant. Plant Physiol 1985; 78:34-40; PMID:16664203; http://dx.doi.org/10.1104/pp.78. 1.34 5. Barbulova A, Rogato A, D’Apuzzo E, Omrane S, Chiurazzi M. Differential effects of combined N sources on early steps of the Nod factor-dependent transduction pathway in Lotus japonicus. Mol PlantMicrobe Interact 2007; 20:994-1003; PMID:17722702; http://dx.doi.org/10.1094/MPMI20-8-0994 6. Searle IR, Men AE, Laniya TS, Buzas DM, IturbeOrmaetxe I, Carroll BJ, Gresshoff PM. Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase. Science 2003; 299:109-12; PMID:12411574; http://dx.doi.org/10.1126/science. 1077937 7. Schnabel E, Journet EP, de Carvalho-Niebel F, Duc G, Frugoli J. The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length. Plant Mol

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Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This research was supported by Grant-in-Aid for JSPS Research Fellows from the Japan Society for the Promotion of Science (no. A2406127 to S.O.), Grants-in-Aid for Scientific Research from the MEXT (no. 22128006 to M.K.).

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Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus.

Nitrate is a major environmental factor in the inhibition of nodulation. In a model legume Lotus japonicus, a CLV1-like receptor kinase, HAR1, mediate...
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