Some species of legumes and nine other flowering plant families form symbioses with bacteria that fix atmospheric nitrogen within specialized plant structures called nodules. How and how often nodulation symbiosis originated has implications for engineering symbiotic nitrogen fixation in non-legume crops. The prevailing hypothesis of a single origin with massive parallel losses has been challenged in a phylogenomic study favoring 16 origins and 10 losses. Nodulation has been assembled once or many times from existing processes (e.g., mycorrhizal symbiosis) and therefore almost nothing about it is truly novel. Because any feature of nodulation can be explained either as divergence from a common origin or as convergence in unrelated taxa, tests are needed that can distinguish whether assembly of homologous components has occurred uniquely or convergently. Much needs to be learned about nodulation symbioses across the proposed independent origins, especially involving the master nodulation transcription factor, Nodule Inception (NIN).

All nitrogen-fixing root nodule symbioses of angiosperms–legume and actinorhizal symbioses–possess a common ancestor. Molecular processes for the induction of root nodules are modulated by phytohormones, as is the case of the first nodulation-related transcription factor NODULE INCEPTION (NIN), whose expression can be induced by exogenous cytokinin in legumes. The process of actinorhizal nodule organogenesis is less well understood. To study the changes exerted by phytohormones on the expression of the orthologs of CYCLOPS, NIN, and NF-YA1 in the actinorhizal host Datisca glomerata, an axenic hydroponic system was established and used to examine the transcriptional responses (RTqPCR) in roots treated with the synthetic cytokinin 6-Benzylaminopurine (BAP), the natural auxin Phenylacetic acid (PAA), and the synthetic auxin 1-Naphthaleneacetic acid (NAA). The model legume Lotus japonicus was used as positive control. Molecular readouts for auxins and cytokinin were established: DgSAUR1 for PAA, DgGH3.1. for NAA, and DgARR9 for BAP. L. japonicus NIN was induced by BAP, PAA, and NAA in a dosage- and time-dependent manner. While expression of D. glomerata NIN2 could not be induced in roots, D. glomerata NIN1 was induced by PAA; this induction was abolished in the presence of exogenous BAP. Furthermore, the induction of DgNIN1 expression by PAA required ethylene and gibberellic acid. This study suggests that while cytokinin signaling is central for cortex-induced nodules of L. japonicus, it acts antagonistically to the induction of nodule primordia of D. glomerata by PAA in the root pericycle.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodulespecific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.

Uptake hydrogenase (Hup) recycles H₂ formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup⁻, while only one Hup⁻  Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a, and 3b according to Søndergaard, Pedersen, and Greening (HydDB: a web tool for hydrogenase classification and analysis. Sci Rep 2016;6:34212. https://doi.org/10.1038/srep34212.); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h, and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H₂ formed by nitrogenase while group 1 h and group 3b are not.

Coriariaceae are a small plant family of 14–17 species and subspecies that currently have a global but disjunct distribution. All species can form root nodules in symbiosis with diazotrophic Frankia cluster-2 strains, which form the earliest divergent symbiotic clade within this bacterial genus. Studies on Frankia cluster-2 mostly have focused on strains occurring in the northern hemisphere. Except for one strain from Papua New Guinea, namely Candidatus Frankia meridionalis Cppng1, no complete genome of Frankia associated with Coriaria occurring in the southern hemisphere has been published thus far, yet the majority of the Coriariaceae species occur here. We present field sampling data of novel Frankia cluster-2 strains, representing two novel species, which are associated with Coriaria arborea and Coriaria sarmentosa in New Zealand, and with Coriaria ruscifolia in Patagonia (Argentina), in addition to identifying Ca. F. meridionalis present in New Zealand. The novel Frankia species were found to be closely related to both Ca. F. meridionalis, and a Frankia species occurring in the Philippines, Taiwan, and Japan. Our data suggest that the different Frankia cluster-2 species diverged early after becoming symbiotic circa 100 million years ago. 

Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1′′→6′)-β-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1′′→6′)-β-d-glucopyranoside, and 3-pentyl 6′-(3-hydroxy3-methylglutaryl)-β-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.