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Legumes form a symbiosis with atmospheric nitrogen (N2 )-fixing soil rhizobia, resulting in new root organs called nodules that enable N2 -fixation. Nodulation is a costly process that is tightly regulated by the host through autoregulation of nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been investigated in Medicago truncatula, for which only rhizobia-induced MtCLE12 and MtCLE13 have been characterised. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. MtCLE35 was found to inhibit nodulation in a SUNN- and RDN1-dependent manner via overexpression analysis. Together, our findings indicate that MtCLE12 and MtCLE13 have a specific role in AON, while MtCLE35 regulates nodule numbers in response to both rhizobia and nitrate. MtCLE34 likely had a similar role to MtCLE35, but its function was lost due to a premature nonsense mutation.

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen-fixing rhizobia bacteria is central to their advantage. This plant-microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.

Cytokinins are important regulators of cell proliferation and differentiation in plant development. Here, a role for this phytohormone group in soybean nodulation is shown through the exogenous application of cytokinins (6-benzylaminopurine, N6-(Δ2-isopentenyl)-adenine and trans-zeatin) via either root drenching or a petiole feeding technique. Overall, nodule numbers were reduced by treatment with high cytokinin concentrations, but increased with lower concentrations. This was especially evident when feeding the solutions directly into the vasculature via petiole feeding. These findings highlight the importance of cytokinin in nodule development. To further investigate the role of cytokinin in controlling nodule numbers, the IPT gene family involved in cytokinin biosynthesis was characterized in soybean. Bioinformatic analyses identified 17 IPT genes in the soybean genome and homeologous duplicate gene partners were subsequently identified including GmIPT5 and GmIPT6, the orthologs of LjIPT3. Expression of GmIPT5 was upregulated in the shoot in response to nodulation, but this was independent of a functional copy of the autoregulation of nodulation (AON) receptor, GmNARK, which suggests it is unlikely to have a role in the negative feedback system called AON. Legumes also control nodule numbers in the presence of soil nitrogen through nitrate-dependent regulation of nodulation, a locally acting pathway in soybean. Upon nitrate treatment to the root, the tandem duplicates GmIPT3 and GmIPT15 were upregulated in expression indicating a role for these genes in the plant's response to soil nitrogen, potentially including the nitrate-dependent regulation of legume nodulation pathway. Additional roles for cytokinin and their IPT biosynthetic genes in nodulation and the control of nodule numbers are discussed.