Brett J Ferguson and Peter M Gresshoff, Centre for Integrative Legume Research, University of Queensland, Australia

Forming nodules and supplying rhizobia with food for nitrogen fixation are energy demanding processes for the host plant. As a result, legumes have developed innate mechanisms to balance their need to acquire nitrogen with their ability to expend energy (reviewed in Ferguson et al. 2010; Reid et al. 2011).

Nitrogen availability: why form nodules when there is plenty of nitrogen available?

When ample nitrogen is available in the soil, legume plants require fewer nodules to meet their nitrogen demands. Accordingly, they have an inbuilt mechanism in the root to detect the nitrogen content of the surrounding soil. Nitrogen-based compounds, such as nitrate, trigger the production of a small, hormone-like peptide signal, called Nitrate-Induced CLE 1 (NIC1) in soybean. NIC1 is predicted to be perceived by the receptor, Nodule Autoregulation Receptor Kinase (NARK). This perception results in the production of a novel inhibitor signal that acts locally in the root to prevent further nodulation events.

Stress: nodulation is a luxury for a stressed plant.

Nodulation is reduced in plants experiencing stress. This likely helps the plant to conserve its resources for combating the stress and for all-important seed development. To date, a number of stress-related factors have been found to inhibit nodule formation locally in the root, including ethylene, salicylic acid and various reactive oxygen species (reviewed in Ferguson and Mathesius 2014). Acidic soil conditions also reduce nodulation, with low pH also causing elevated soil Al³⁺ levels that negatively affect root growth.

Autoregulation of nodulation: too much of a good thing is not good.

Less than 10% of rhizobia infection events result in the formation of a fully-developed nodule. This is largely due to the Autoregulation Of Nodulation (AON) mechanism that the host plant uses to control its nodule numbers (reviewed by Ferguson et al. 2010; Reid et al. 2011). Mutant legume plants lacking a functional AON pathway are unable to regulate their nodule numbers and as a result exhibit a supernodulation phenotype (i.e. they develop an excessive number of nodules, with up to 25,000 per plant scored, Figure 1). When these supernodulating mutants are induced to form nodules, they are typically reduced in stature and often yield about 20 – 30% less, likely a direct result of their resources being used to form excess nodule structures (Figure 1).

The AON process is triggered within hours of rhizobia inoculation. It involves long-distance signalling between the root and shoot, commencing with the production of a root-derived signal (Figure 2). Recent work has indicated that this signal is a CLE peptide hormone, highly similar to NIC1. In soybean, two candidate CLE peptide signals have been identified and are called Rhizobia Induced CLE1 (RIC1) and RIC2. The use of grafting and over-expression experiments demonstrated that RIC1 and RIC2 travel to the shoot, likely via the xylem. They are thought to be perceived in the shoot by the same receptor kinase that detects NIC1 in the root, namely NARK, possibly in combination with other receptors, such as CLAVATA2 and KLAVIER. Indeed, this has now been confirmed for orthologous peptides of Lotus japonicus, called LjCLE-RS1 and 2, which travel in the xylem and are perceived by the orthologue of NARK in L. japonicus, HAR1 (Okamoto et al., 2013). The peptides are post-translationally modified with three arabinose sugars attached to a central proline residue, likely by an arabinotransferase encoded by the NOD3/RDN1 gene in Pisum sativum and Medicago truncatula (Ogawa-Ohnishi et al. 2013). Key domains and amino acid residues of soybean RIC1 that are required for effective suppression of nodulation have also been identified. Perception of either RIC1 or RIC2 triggers the production of a new signal, called the Shoot Derived Inhibitor (SDI). SDI is subsequently transported down to the root where it acts to inhibit further nodulation events (Figure 2).

Although it has not yet been identified, SDI is reported to be a small, heat-stable, Nod factor- and NARK-dependent molecule that is not likely an RNA or protein (Lin et al. 2010) and has been recently been proposed to be the classical phytohormone, cytokinin (Sasaki et al. 2014). Additional factors acting downstream of SDI include Too Much Love, a Kelch-repeat transcription factor whose role in the AON process is yet to be fully defined.

An interesting finding is that of Wang et al. (2014), who suggest that the AON pathway may involve a microRNA after the tentative cytokinin signal. Specifically miR172c was shown to negatively regulate the transcript abundance of a gene (GmNNC1) encoding a transcription factor, being part of the AP2 family. GmNNC1 negatively targets the early nodulin gene ENOD40, needed for nodulation progress in soybean, Medicago truncatula, and Lotus japonicus. It now appears critical to connect the function of a peptide-activated receptor (GmNARK) with the reported cytokinin signal and the subsequent negative regulation cascade described by Wang et al. (2014).

References

Ferguson BJ, Indrasumunar A, Hayashi S et al. (2010) Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol 52: 61-76

Ferguson BJ, Mathesius U (2014) Phytohormone regulation of legume-rhizobia interactions. J Chem Ecol 40: 770-790

Lin Y-H, Ferguson BJ, Kereszt A, Gresshoff PM (2010) Suppression of hypernodulation in soybean by a leaf-extracted, NARK- and Nod factor-dependent small molecular fraction. New Phytol 185: 1074-1086

Okamoto S, Shinohara H, Mori T et al. (2013) Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase. Nature Comms 4, doi:10.1038/ncomms3191

Ogawa-Ohnishi M, Matsushita W, Matsubayashi Y (2013). Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana. Nature Chem Biol 9: 726-730

Reid DE, Ferguson BJ, Hayashi S et al. (2011) Molecular mechanisms controlling legume autoregulation of nodulation. Ann Botany 108: 789-795

Sasaki T, Suzaki T, Soyano T et al. (2014). Shoot-derived cytokinins systemically regulate root nodulation. Nature Comms 5, doi:10.1038/ncomms5983.

Wang Y, Wang L, Zou Y et al. (2014) Soybean miR172c targets the repressive AP2 transcription factor GmNNC1 to activate GmENOD40 expression and regulate nodule initiation. Plant Cell 26: 4782–4801