Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules.

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing "ecometabolome" approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

Identification of potential transcriptional regulators of actinorhizal symbioses in Casuarina glauca and Alnus glutinosa.

BACKGROUND: Trees belonging to the Casuarinaceae and Betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. These functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus Frankia. However, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. The aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses. RESULTS: We identified 202 putative transcription factors by in silico analysis in 40 families in Casuarina glauca (Casuarinaceae) and 195 in 35 families in Alnus glutinosa (Betulaceae) EST databases. Based on published transcriptome datasets and quantitative PCR analysis, we found that 39% and 26% of these transcription factors were regulated during C. glauca and A. glutinosa-Frankia interactions, respectively. Phylogenetic studies confirmed the presence of common key transcription factors such as NSP, NF-YA and ERN-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. We also identified an actinorhizal-specific transcription factor belonging to the zinc finger C1-2i subfamily we named CgZF1 in C. glauca and AgZF1 in A. glutinosa. CONCLUSIONS: We identified putative nodulation-associated transcription factors with particular emphasis on members of the GRAS, NF-YA, ERF and C2H2 families. Interestingly, comparison of the non-legume and legume TF with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific C2H2 TF that could be specifically involved in actinorhizal symbioses. In silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to Frankia infection.