Zinc mediates control of nitrogen fixation via transcription factor filamentation.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Constitutive activation of a nuclear-localized calcium channel complex in Medicago truncatula.

Nuclear Ca2+ oscillations allow symbiosis signaling, facilitating plant recognition of beneficial microsymbionts, nitrogen-fixing rhizobia, and nutrient-capturing arbuscular mycorrhizal fungi. Two classes of channels, DMI1 and CNGC15, in a complex on the nuclear membrane, coordinate symbiotic Ca2+ oscillations. However, the mechanism of Ca2+ signature generation is unknown. Here, we demonstrate spontaneous activation of this channel complex, through gain-of-function mutations in DMI1, leading to spontaneous nuclear Ca2+ oscillations and spontaneous nodulation, in a CNGC15-dependent manner. The mutations destabilize a hydrogen-bond or salt-bridge network between two RCK domains, with the resultant structural changes, alongside DMI1 cation permeability, activating the channel complex. This channel complex was reconstituted in human HEK293T cell lines, with the resultant calcium influx enhanced by autoactivated DMI1 and CNGC15s. Our results demonstrate the mode of activation of this nuclear channel complex, show that DMI1 and CNGC15 are sufficient to create oscillatory Ca2+ signals, and provide insights into its native mode of induction.

SCARN a Novel Class of SCAR Protein That Is Required for Root-Hair Infection during Legume Nodulation.

Rhizobial infection of legume root hairs requires a rearrangement of the actin cytoskeleton to enable the establishment of plant-made infection structures called infection threads. In the SCAR/WAVE (Suppressor of cAMP receptor defect/WASP family verpolin homologous protein) actin regulatory complex, the conserved N-terminal domains of SCAR proteins interact with other components of the SCAR/WAVE complex. The conserved C-terminal domains of SCAR proteins bind to and activate the actin-related protein 2/3 (ARP2/3) complex, which can bind to actin filaments catalyzing new actin filament formation by nucleating actin branching. We have identified, SCARN (SCAR-Nodulation), a gene required for root hair infection of Lotus japonicus by Mesorhizobium loti. Although the SCARN protein is related to Arabidopsis thaliana SCAR2 and SCAR4, it belongs to a distinct legume-sub clade. We identified other SCARN-like proteins in legumes and phylogeny analyses suggested that SCARN may have arisen from a gene duplication and acquired specialized functions in root nodule symbiosis. Mutation of SCARN reduced formation of infection-threads and their extension into the root cortex and slightly reduced root-hair length. Surprisingly two of the scarn mutants showed constitutive branching of root hairs in uninoculated plants. However we observed no effect of scarn mutations on trichome development or on the early actin cytoskeletal accumulation that is normally seen in root hair tips shortly after M. loti inoculation, distinguishing them from other symbiosis mutations affecting actin nucleation. The C-terminal domain of SCARN binds to ARPC3 and ectopic expression of the N-terminal SCAR-homology domain (but not the full length protein) inhibited nodulation. In addition, we found that SCARN expression is enhanced by M. loti in epidermal cells and that this is directly regulated by the NODULE INCEPTION (NIN) transcription factor.

Staying hungry: a roadmap to harnessing central regulators of symbiotic nitrogen fixation under fluctuating nitrogen availability.

Legumes have evolved specific inventions to enhance nitrogen (N) acquisition by establishing symbiotic interactions with N-fixing rhizobial bacteria. Because symbiotic N fixation is energetically costly, legumes have developed sophisticated mechanisms to ensure carbon-nitrogen balance, in a variable environment, both locally and at the whole plant level, by monitoring nodule number, nodule development, and nodular nitrogenase activity, as well as controlling nodule senescence. Studies of the autoregulation of nodulation and regulation of nodulation by nodule inception (NIN) and NIN-LIKE PROTEINs (NLPs) have provided great insights into the genetic mechanisms underlying the nitrate-induced regulation of root nodulation for adapting to N availability in the rhizosphere. However, many aspects of N-induced pleiotropic regulation remain to be fully explained, such as N-triggered senescence in mature nodules. Wang et al. determined that this process is governed by a transcriptional network regulated by NAC-type transcription factors. Characterization and dissection of these soybean nitrogen-associated NAPs (SNAPs) transcription factor-mastered networks have yielded a roadmap for exploring how legumes rewire nodule functions across a range of N levels, laying the foundation for enhancing the growth of N-deprived crops in agricultural settings.

Nitrate restricts nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus.

Legumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application rescues nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.

NLP1 reciprocally regulates nitrate inhibition of nodulation through SUNN-CRA2 signaling in Medicago truncatula.

Most legume plants can associate with diazotrophic soil bacteria called rhizobia, resulting in new root organs called nodules that enable N2 fixation. Nodulation is an energy-consuming process, and nodule number is tightly regulated by independent systemic signaling pathways controlled by CLE/SUNN and CEP/CRA2. Moreover, nitrate inhibits legume nodulation via local and systemic regulatory pathways. In Medicago truncatula, NLP1 plays important roles in nitrate-induced inhibition of nodulation, but the relationship between systemic and local pathways in mediating nodulation inhibition by nitrate is poorly understood. In this study, we found that nitrate induces CLE35 expression in an NLP1-dependent manner and that NLP1 binds directly to the CLE35 promoter to activate its expression. Grafting experiments revealed that the systemic control of nodule number involves negative regulation by SUNN and positive regulation by CRA2 in the shoot, and that NLP1's control of the inhibition of rhizobial infection, nodule development, and nitrogenase activity in response to nitrate is determined by the root. Unexpectedly, grafting experiments showed that loss of CRA2 in the root increases nodule number at inhibitory nitrate levels, probably because of CEP1/2 upregulation in the cra2 mutants, suggesting that CRA2 exerts active negative feedback regulation in the root.

No Home without Hormones: How Plant Hormones Control Legume Nodule Organogenesis.

The establishment of symbiotic nitrogen fixation requires the coordination of both nodule development and infection events. Despite the evolution of a variety of anatomical structures, nodule organs serve a common purpose in establishing a localized area that facilitates efficient nitrogen fixation. As in all plant developmental processes, the establishment of a new nodule organ is regulated by plant hormones. During nodule initiation, regulation of plant hormone signaling is one of the major targets of symbiotic signaling. We review the role of major developmental hormones in the initiation of the nodule organ and argue that the manipulation of plant hormones is a key requirement for engineering nitrogen fixation in non-legumes as the basis for improved food security and sustainability.

Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence.

Lotus japonicus is a herbaceous perennial legume that has been used extensively as a genetically tractable model system for deciphering the molecular genetics of symbiotic nitrogen fixation. Our aim is to improve the L. japonicus reference genome sequence, which has so far been based on Sanger and Illumina sequencing reads from the L. japonicus accession MG-20 and contained a large fraction of unanchored contigs. Here, we use long PacBio reads from L. japonicus Gifu combined with Hi-C data and new high-density genetic maps to generate a high-quality chromosome-scale reference genome assembly for L. japonicus. The assembly comprises 554 megabases of which 549 were assigned to six pseudomolecules that appear complete with telomeric repeats at their extremes and large centromeric regions with low gene density. The new L. japonicus Gifu reference genome and associated expression data represent valuable resources for legume functional and comparative genomics. Here, we provide a first example by showing that the symbiotic islands recently described in Medicago truncatula do not appear to be conserved in L. japonicus.

NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula.

Legume plants can assimilate inorganic nitrogen and have access to fixed nitrogen through symbiotic interaction with diazotrophic bacteria called rhizobia. Symbiotic nitrogen fixation is an energy-consuming process and is strongly inhibited when sufficient levels of fixed nitrogen are available, but the molecular mechanisms governing this regulation are largely unknown. The transcription factor nodule inception (NIN) is strictly required for nodulation and belongs to a family of NIN-like proteins (NLPs), which have been implicated in the regulation of nitrogen homeostasis in Arabidopsis. Here, we show that mutation or downregulation of NLP genes prevents nitrate inhibition of infection, nodule formation and nitrogen fixation. We find that NIN and NLPs physically interact through their carboxy-terminal PB1 domains. Furthermore, we find that NLP1 is required for the expression of nitrate-responsive genes and that nitrate triggers NLP1 re-localization from the cytosol to the nucleus. Finally, we show that NLP1 can suppress NIN activation of CRE1 expression in Nicotiana benthamiana and Medicago truncatula. Our findings highlight a central role for NLPs in the suppression of nodulation by nitrate.

Author Correction: NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula.

In the version of this Article originally published, the name of author Zhenpeng Luo was incorrectly listed as Zhenpeng Luo Luo. Additionally, nitrate in Fig. 6b was incorrectly shown as 'NO3+' rather than 'NO3-'. This has now been amended.