Two types of nitrogen-fixing root nodule symbioses are known, rhizobial and actinorhizal symbioses. The latter involve plants of three orders, Fagales, Rosales, and Cucurbitales. To understand the diversity of plant symbiotic adaptation, we compared the nodule transcriptomes of Datisca glomerata (Datiscaceae, Cucurbitales) and Ceanothus thyrsiflorus (Rhamnaceae, Rosales); both species are nodulated by members of the uncultured Frankia clade, cluster II. The analysis focused on various features. In both species, the expression of orthologs of legume Nod factor receptor genes was elevated in nodules compared to roots. Since arginine has been postulated as export form of fixed nitrogen from symbiotic Frankia in nodules of D. glomerata, the question was whether the nitrogen metabolism was similar in nodules of C. thyrsiflorus. Analysis of the expression levels of key genes encoding enzymes involved in arginine metabolism revealed up-regulation of arginine catabolism, but no up-regulation of arginine biosynthesis, in nodules compared to roots of D. glomerata, while arginine degradation was not upregulated in nodules of C. thyrsiflorus. This new information corroborated an arginine-based metabolic exchange between host and microsymbiont for D. glomerata, but not for C. thyrsiflorus. Oxygen protection systems for nitrogenase differ dramatically between both species. Analysis of the antioxidant system suggested that the system in the nodules of D. glomerata leads to greater oxidative stress than the one in the nodules of C. thyrsiflorus, while no differences were found for the defense against nitrosative stress. However, induction of nitrite reductase in nodules of C. thyrsiflorus indicated that here, nitrite produced from nitric oxide had to be detoxified. Additional shared features were identified: genes encoding enzymes involved in thiamine biosynthesis were found to be upregulated in the nodules of both species. Orthologous nodule-specific subtilisin-like proteases that have been linked to the infection process in actinorhizal Fagales, were also upregulated in the nodules of D. glomerata and C. thyrsiflorus. Nodule-specific defensin genes known from actinorhizal Fagales and Cucurbitales, were also found in C. thyrsiflorus. In summary, the results underline the variability of nodule metabolism in different groups of symbiotic plants while pointing at conserved features involved in the infection process.

Two types of nitrogen-fixing root nodules are known, legume nodules and actinorhizal nodules. During the induction of nitrogen-fixing root nodules, phytohormones are involved in regulating gene expression to shape essential processes. One example is the expression of the early nodulin NODULE INCEPTION (NIN), which in the model legume Lotus japonicus is induced by cytokinin. In order to develop a bioassay for a quick and superficial analysis how phytohormones affect the expression of nodulation-related genes in different root nodule-forming plants, an axenic liquid culture system was established. The bioassay was used to examine responses in roots of the actinorhizal plant Datisca glomerata to phytohormones using quantitative RT-PCR. The synthetic cytokinin 6-Benzylaminopurine (BAP), the natural auxin phenyl acetic acid (PAA), and the synthetic auxin 1-Naphthaleneacetic Acid (NAA) were used. Marker genes for auxins and cytokinin responses were identified. PAA induced the expression of DgSAUR1, whereas NAA induced DgGH3.1.

Induction of Lotus japonicus NIN was analysed for proof of concept, and the bioassay showed induction by cytokinin and auxin (NAA). D. glomerata NIN1 was induced by PAA, NAA, and more weakly by BAP. The gene encoding a transcription factor that activates NIN expression, CYCLOPS, was induced by PAA and NAA. To see whether induction of NIN1 expression led to the activation of target genes of NIN, orthologs of the transcription factors NF-YA1, NSP1 and NSP2, and of ERN1 were identified in D. glomerata and the transcription of the corresponding genes was analysed for regulation by BAP, PAA or NAA. Induction by PAA was found for all genes examined. Additionally, BAP and PAA were used individually or in combination with antagonists of ethylene or gibberellin biosynthesis, respectively. Both antagonists abolished the induction of NIN by BAP or PAA; furthermore, no induction of NIN was found when BAP and PAA were applied together.

Altogether, these results show that the bioassay system can be used to identify target genes of NIN in legumes and actinorhizal plants for further analysis of the role of phytohormones in nodule induction.

Several types of cysteine-rich peptides are involved in the interaction between plants and microorganisms, e.g., anti-bacterial and anti-fungal defensins, characterized by eight conserved cysteine residues, or nodule-specific cysteine-rich peptides that manipulate microsymbiont development in legume nodules, containing five to six conserved cysteine residues. Nodule-specific defensins are found in all actinorhizal plants of Fagales, Rosales as well as Cucurbitales, and a defensin from Alnus glutinosa nodules was shown to affect membrane integrity of a compatible Frankia strain and cause leakage of amino acids (Carro et al., 2015).

Nodules of Datisca glomerata (Datiscaceae. Cucurbitales) contain a gene family encoding nodule-specific defensins with an acidic C-terminal domain. The member expressed at the highest levels, DgDef1, was expressed in young infected cells and, transiently, above the nodule lobe meristem. Neither the complete protein excluding the N-terminal signal peptide produced in Pichia pastoris, nor the synthetically produced cysteine-rich domain had not effect on the growth of Gram-positive Streptomyces coelicolor. However, the cysteine-rich domain alone had a cytotoxic effect on Gram-negative E. coli K-12 and on Sinorhizobium meliloti 1021 with an ID₅₀ of 20.8 µM. RNAseq analysis of Sm1021 cultures treated with this domain, compared to an untreated control, showed a strong effect on the expression of genes encoding transporters, e.g., a dicarboxylate uptake system, consistent with the fact that treated Sm1021 cells had leaky membrane as denoted by propidium iodide staining. However, the expression of the cell cycle regulator ctrA was not affected, suggesting that DgDef1 did not affect the ploidity of Sm1021. Phylogenetic analysis of the cysteine-rich domains showed that legume NCRs and actinorhizal nodule-specific defensins go back to a common ancestor.

Two types of root nodule symbioses between nitrogen-fixing soil bacteria and higher plants are known, rhizobia/legume symbioses and the symbiosis between soil actinobacteria of the genus Frankia and more than 200 species from three different orders, collectively called actinorhizal plants. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen. Based on studies with bacterial mutants, it is known that rhizobia are supplied with dicarboxylates, specifically malate, but no legume malate exporters involved in the symbiosis have been identified thus far. For Frankia, a malate exporter from the NPF transporter family that locates to the perisymbiont membrane was identified in Alnus glutinosa (AgDCAT1; Jeong et al., 2004).   

In this study, the substrate specificity of two homologs of AgDCAT1 from nodules of two different actinorhizal hosts was analysed: CgDCAT1 from Casuarina glauca (Casuarinaceae, Fagales), a close relative of A. glutinosa, and DgDCAT1 from Datisca glomerata (Datiscaceae, Cucurbitales). A Multidrug And Toxic compound Extrusion (MATE) type protein from D. glomerata nodules (DgMATE1) was included in the analysis. The transcription of the corresponding genes of all three proteins was induced strongly in nodules compared to roots. Experiments using Xenopus laevis oocytes pre-loaded with ¹³C-labeled metabolites showed that all three proteins represent citrate exporters. Studies on the levels of citrate cycle intermediates showed that both citrate and malate were present at dramatically higher levels that 2‑oxoglutarate, succinate and fumate in roots and nodules of C. glauca as well as D. glomerata. The Casuarina-nodulating strain Frankia casuarinae CcI3 grew equally well on malate or citrate. In short, the results of this study led to the conclusion that intracellular Frankia bacteria are fed with citrate, not malate, in nodules of C. glauca and D. glomerata.

AgDCAT1 and CgDCAT1 represent orthologs, but DgDCAT1 maps to another subclade of the NPF family, showing that the NPF carboxylate exporters in actinorhizal nodules are polyphyletic. DgMATE1 represents a member of the detoxification (DTX) subgroup that seems to have been recruited for supplying Frankia with citrate. The data suggest that in different symbiotic lineages, carbon source exporters for the symbiosis were recruited from different transporter families independently.