Symbiotic Performance of Diverse Frankia Strains on Salt-Stressed Casuarina glauca and Casuarina equisetifolia Plants

Symbiotic nitrogen-fixing associations between Casuarina trees and the actinobacteria Frankia are widely used in agroforestry in particular for salinized land reclamation. The aim of this study was to analyze the effects of salinity on the establishment of the actinorhizal symbiosis between C. glauca and two contrasting Frankia strains (salt sensitive; CcI3 vs. salt tolerant; CeD) and the role of these isolates in the salt tolerance of C. glauca and C. equisetifolia plants. We show that the number of root nodules decreased with increasing salinity levels in both plants inoculated with CcI3 and CeD. Nodule formation did not occur in seedlings inoculated with CcI3 and CeD, at NaCl concentrations above 100 and 200 mM, respectively. Salinity also affected the early deformation of plant root hairs and reduced their number and size. In addition, expression of symbiotic marker Cg12 gene, which codes for a subtilase, was reduced at 50 mM NaCl. These data suggest that the reduction of nodulation in C. glauca under salt stress is in part due to inhibition of early mechanisms of infection. We also show that prior inoculation of C. glauca and C. equisetifolia with Frankia strains CcI3 and CeD significantly improved plant height, dry biomass, chlorophyll and proline contents at all levels of salinity tested, depending on the Casuarina-Frankia association. There was no correlation between in vitro salt tolerance of Frankia strains and efficiency in planta under salt-stressed conditions. Our results strongly indicate that increased N nutrition, photosynthesis potential and proline accumulation are important factors responsible for salt tolerance of nodulated C. glauca and C. equisetifolia

Actinorhizal Signaling Molecules: Frankia Root Hair Deforming Factor Shares Properties With NIN Inducing Factor

Actinorhizal plants are able to establish a symbiotic relationship with Frankia bacteria leading to the formation of root nodules. The symbiotic interaction starts with the exchange of symbiotic signals in the soil between the plant and the bacteria. This molecular dialog involves signaling molecules that are responsible for the specific recognition of the plant host and its endosymbiont. Here we studied two factors potentially involved in signaling between Frankia casuarinae and its actinorhizal host Casuarina glauca: (1) the Root Hair Deforming Factor (CgRHDF) detected using a test based on the characteristic deformation of C. glauca root hairs inoculated with F. casuarinae and (2) a NIN activating factor (CgNINA) which is able to activate the expression of CgNIN, a symbiotic gene expressed during preinfection stages of root hair development. We showed that CgRHDF and CgNINA corresponded to small thermoresistant molecules. Both factors were also hydrophilic and resistant to a chitinase digestion indicating structural differences from rhizobial Nod factors (NFs) or mycorrhizal Myc-LCOs. We also investigated the presence of CgNINA and CgRHDF in 16 Frankia strains representative of Frankia diversity. High levels of root hair deformation (RHD) and activation of ProCgNIN were detected for Casuarina-infective strains from clade Ic and closely related strains from clade Ia unable to nodulate C. glauca. Lower levels were present for distantly related strains belonging to clade III. No CgRHDF or CgNINA could be detected for Frankia coriariae (Clade II) or for uninfective strains from clade IV

New Insights on plant salt tolerance mechanisms and their potential use for breeding

Soil salinization is a major threat to agriculture in arid and semi-arid regions, where water scarcity and inadequate drainage of irrigated lands severely reduce crop yield. Salt accumulation inhibits plant growth and reduces the ability to uptake water and nutrients, leading to osmotic or water-deficit stress. Salt is also causing injury of the young photosynthetic leaves and acceleration of their senescence, as the Na+ cation is toxic when accumulating in cell cytosol resulting in ionic imbalance and toxicity of transpiring leaves. To cope with salt stress, plants have evolved mainly two types of tolerance mechanisms based on either limiting the entry of salt by the roots, or controlling its concentration and distribution. Understanding the overall control of Na+ accumulation and functional studies of genes involved in transport processes, will provide a new opportunity to improve the salinity tolerance of plants relevant to food security in arid regions. A better understanding of these tolerance mechanisms can be used to breed crops with improved yield performance under salinity stress. Moreover, associations of cultures with nitrogen-fixing bactéria and arbuscular mycorrhizal fungi could serve as an alternative and sustainable strategy to increase crop yields in salt affected fields

New Insights on Plant Salt Tolerance Mechanisms and Their Potential Use for Breeding

International audienceSoil salinization is a major threat to agriculture in arid and semi-arid regions, where water scarcity and inadequate drainage of irrigated lands severely reduce crop yield. Salt accumulation inhibits plant growth and reduces the ability to uptake water and nutrients, leading to osmotic or water-deficit stress. Salt is also causing injury of the young photosynthetic leaves and acceleration of their senescence, as the Na + cation is toxic when accumulating in cell cytosol resulting in ionic imbalance and toxicity of transpiring leaves. To cope with salt stress, plants have evolved mainly two types of tolerance mechanisms based on either limiting the entry of salt by the roots, or controlling its concentration and distribution. Understanding the overall control of Na + accumulation and functional studies of genes involved in transport processes, will provide a new opportunity to improve the salinity tolerance of plants relevant to food security in arid regions. A better understanding of these tolerance mechanisms can be used to breed crops with improved yield performance under salinity stress. Moreover, associations of cultures with nitrogen-fixing bacteria and arbuscular mycorrhizal fungi could serve as an alternative and sustainable strategy to increase crop yields in salt-affected fields

Symbiotic ability of diverse Frankia strains on Casuarina glauca plants in hydroponic conditions

International audienceFrankia is a soil actinomycete that forms nitrogen-fixing root nodule symbioses with eight angiosperms families including Casuarinaceae. Knowledge on symbiotic performance of several isolated strains with Casuarina species is limited. In this study, we characterized a collection of Frankia strains based on their growth kinetics and their symbiotic ability with Casuarina glauca specie. Results showed that Frankia strains Allo2, CcI3, CeD and Cg70.9 do not exhibit the same symbiotic ability both for their infectivity and effectiveness towards Casuarina glauca. All strains were able to infect and improve C. glauca plants growth. Frankia isolate CcI3 better improved plants height while CeD and Cg70.9 strains formed more nodules on inoculated plants roots. However, there is no correlation between the number of nodules formed and the effectiveness of strains. These preliminary results give ideas on which Frankia strains to use for obtaining an efficient symbiosis with C. glauca. However, others plant species and growth conditions should be tested for a longer period to better optimize the use of Frankia in reforestation programs

Roles of Arbuscular Mycorrhizal Fungi on Plant Growth and Performance: Importance in Biotic and Abiotic Stressed Regulation

International audienceArbuscular mycorrhizal fungi (AMF) establish symbiotic associations with most terrestrial plants. These soil microorganisms enhance the plant's nutrient uptake by extending the root absorbing area. In return, the symbiont receives plant carbohydrates for the completion of its life cycle. AMF also helps plants to cope with biotic and abiotic stresses such as salinity, drought, extreme temperature, heavy metal, diseases, and pathogens. For abiotic stresses, the mechanisms of adaptation of AMF to these stresses are generally linked to increased hydromineral nutrition, ion selectivity, gene regulation, production of osmolytes, and the synthesis of phytohormones and antioxidants. Regarding the biotic stresses, AMF are involved in pathogen resistance including competition for colonization sites and improvement of the plant's defense system. Furthermore, AMF have a positive impact on ecosystems. They improve the quality of soil aggregation, drive the structure of plant and bacteria communities, and enhance ecosystem stability. Thus, a plant colonized by AMF will use more of these adaptation mechanisms compared to a plant without mycorrhizae. In this review, we present the contribution of AMF on plant growth and performance in stressed environments

Signalling in actinorhizal root nodule symbioses

International audiencePlants able to establish a nitrogen-fixing root nodule symbiosis with the actinobacterium Frankia are called actinorhizal. These interactions lead to the formation of new root organs, called actinorhizal nodules, where the bacteria are hosted intracellularly and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. Like other symbiotic interactions, actinorhizal nodulation involves elaborate signalling between both partners of the symbiosis, leading to specific recognition between the plant and its compatible microbial partner, its accommodation inside plant cells and the development of functional root nodules. Actinorhizal nodulation shares many features with rhizobial nodulation but our knowledge on the molecular mechanisms involved in actinorhizal nodulation remains very scarce. However recent technical achievements for several actinorhizal species are allowing major discoveries in this field. In this review, we provide an outline on signalling molecules involved at different stages of actinorhizal nodule formation and the corresponding signalling pathways and gene networks

Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees

International audienceBackground: Soil 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.Results: Salt-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.Conclusion: Genetic 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

Selection of arbuscular mycorrhizal fungal strains to improve <em>Casuarina equisetifolia</em> L. and <em>Casuarina glauca</em> Sieb. tolerance to salinity

International audienceContext Land salinization is a serious problem worldwide that mainly leads to soil degradation and reduces crop productivity. These degraded areas could be rehabilitated by planting salt-tolerant species like Casuarina glauca Sieb. and Casuarina equisetifolia L. These are pioneer plants, able to form symbiotic associations with arbuscular mycorrhizal fungi (AMF), ectomycorrhizal fungi (EMF), and nitrogen-fixing bacteria. Aims The aim of this study was to select the highest salt-tolerant combination of Casuarina/ AMF that can be used for the rehabilitation of lands degraded by salinity. Methods C. equisetifolia and C. glauca were grown in sandy sterile soil in the greenhouse and inoculated separately with Rhizophagus fasciculatus (Thaxt.) C. Walker & A. Schussler, Rhizophagus aggregatus (N.C. Schenck & G.S. Sm.) C. Walker, and Rhizophagus intraradices (N.C. Schenck & G.S. Sm.) C. Walker & A. Schussler. After confirming the establishment of a symbiosis, the plants were watered with gradually increasing concentrations of saline solution. After harvest, size and biomass of the seedlings, root colonization by AMF, and AMF metabolic activities were evaluated. Results A larger growth was obtained in the two species when the individuals were inoculated with R. fasciculatus. Root colonization rates did not differ among fungal species, but fungal metabolic activities were higher in mycorrhizal roots of C. glauca plants inoculated with R. fasciculatus. Conclusion Among the three mycorrhizal fungi, R. fasciculatus was more efficient in association with Casuarinaceae species under salt stress. Our results suggest that selection of appropriate fungal strains is crucial to improve plant performance in saline soils

Use of Frankia and Actinorhizal Plants for Degraded Lands Reclamation

Degraded lands are defined by soils that have lost primary productivity due to abiotic or biotic stresses. Among the abiotic stresses, drought, salinity, and heavy metals are the main threats in tropical areas. These stresses affect plant growth and reduce their productivity. Nitrogen-fixing plants such as actinorhizal species that are able to grow in poor and disturbed soils are widely planted for the reclamation of such degraded lands. It has been reported that association of soil microbes especially the nitrogen-fixing bacteria Frankia with these actinorhizal plants can mitigate the adverse effects of abiotic and biotic stresses. Inoculation of actinorhizal plants with Frankia significantly improves plant growth, biomass, shoot and root N content, and survival rate after transplanting in fields. However, the success of establishment of actinorhizal plantation in degraded sites depends upon the choice of effective strains of Frankia. Studies related to the beneficial role of Frankia on the establishment of actinorhizal plants in degraded soils are scarce. In this review, we describe some examples of the use of Frankia inoculation to improve actinorhizal plant performances in harsh conditions for reclamation of degraded lands