Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus

Abstract

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.

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

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 root-to-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 before^8,9 (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 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.

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 = 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.

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 signaling system is shared between nitrate inhibition and autoregulation of nodulation.

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.

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 and nitrate responses of root systems. Therefore, studies on nitrate inhibition of nodulation could provide insights that may help understand how legumes have established long-distance autoregulation of nodulation.

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.).