BackgroundSoil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance.ResultsSalt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the salt-tolerant (CcI6) and the salt-sensitive (CcI3) strains, respectively.ConclusionGenetic differences between salt-tolerant and salt-sensitive Frankia strains isolated from Casuarina were identified. Transcriptome and proteome profiling of a salt-tolerant strain was used to determine molecular differences correlated with differential salt-tolerance and several candidate genes were identified. Mechanisms involving transcriptional and translational regulation, cell envelop remodeling, and previously uncharacterized proteins appear to be important for salt tolerance. Physiological and mutational analyses will further shed light on the molecular mechanism of salt tolerance in Casuarina associated Frankia isolates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-4056-0) contains supplementary material, which is available to authorized users.

Casuarina glauca (Casuarinaceae) is an important introduced tree species in Egypt, valued for windbreaks, land stabilization, and soil improvement associated with actinomycete-induced root nodules that fix atmospheric nitrogen. A strain of Frankia designated HFPCgI4 was isolated from root nodules collected in Egypt and its characteristics assessed both in pure culture and in symbiosis. Strain CgI4 grows well in synthetic nutrient medium with added NH4+ or, in the absence of combined N in the medium, forms vesicles and fixes dinitrogen adequate for growth. Hyphae, vesicles, sporangia, and spores characteristic of the genus Frankia were observed. This strain shows spontaneous spore release when grown in media lacking N. When tested for infectivity on actinorhizal host plants grown in unaerated water culture, CgI4 nodulates several species of Casuarina that fix atmospheric dinitrogen. Other genera in the Casuarinaceae, namely, Allocasuarina and Gymnostoma, were not nodulated under these conditions. Species of the genus Myrica and Comptonia peregrina (Myricaceae) were effectively nodulated by CgI4. The isolate may have use as an inoculant for forest plantations using species of Casuarina sensu stricta.

Graphical abstract

Casuarina glauca is a fast-growing multipurpose tree belonging to the Casuarinaceae family and native to Australia. It requires limited use of chemical fertilizers due to the symbiotic association with the nitrogen-fixing actinomycete Frankia and with mycorrhizal fungi, which help improve phosphorous and water uptake by the root system. C. glauca can grow in difficult sites, colonize eroded lands and improve their fertility, thereby enabling the subsequent growth of more demanding plant species. As a result, this tree is increasingly used for reforestation and reclamation of degraded lands in tropical and subtropical areas such as China and Egypt. Many tools have been developed in recent years to explore the molecular basis of the interaction between Frankia and C. glauca. These tools include in vitro culture of the host and genetic transformation with Agrobacterium, genome sequencing of Frankia and related studies, isolation of plant symbiotic genes combined with functional analyses (including knock-down expression based on RNA interference), and transcriptome analyses of roots inoculated with Frankia or Rhizophagus irregularis. These efforts have been fruitful since recent results established that many common molecular mechanisms regulate the nodulation process in actinorhizal plants and legumes, thus providing new insights into the evolution of nitrogen-fixing symbioses.

Actinorhizal plants are able to overcome saline soils and reclaim land. Frankia sp strain CcI6 was isolated from nodules of Casuarina cunninghamiana found in Egypt. Phylogenetic analysis of Frankia sp. strain CcI6 revealed that the strain is closely related to Frankia sp. strain CcI3. The strain displays an elevated level of NaCl tolerance. Vesicle production and nitrogenase activity were also influenced by NaCl.