An overview of Parafrankia (Nod+/Fix+) and Pseudofrankia (Nod+/Fix-) interactions through genome mining and experimental modeling in co-culture and co-inoculation of Elaeagnus angustifolia.

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix- phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogen-depleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. IMPORTANCE: While Pseudofrankia strains typically lack the common traits of ability to nodulate the host plant (Nod-) and/or fix nitrogen (Fix-), they are still recovered from actinorhizal nodules. The enigmatic question of how and why these unconventional strains establish themselves within nodule tissue, thriving either alongside symbiotic strains (Nod+/Fix+) or independently, while considering potential metabolic costs to the host plant, remains a perplexing puzzle. This study endeavors to unravel the competitive dynamics between Pseudofrankia inefficax strain EuI1c (Nod+/Fix-) and Parafrankia strain EU1Nf (Nod+/Fix+) through a comprehensive exploration of genomic data and empirical modeling, conducted both in controlled laboratory settings and within the host plant environment.

Cyclopeptide Alkaloids from Discaria chacaye (Rhamnaceae) as Result of Symbiosis with Frankia (Actinomycetales).

Cyclopeptide alkaloids with different biological activities are present in plants of the family Rhamnaceae. Plants of this family grow in a symbiotic relationship with aerobic Gram-positive actinomycetes belonging to the genus Frankia. This goal of this research was a study of the comparative profile of alkaloids present in Discaria chacaye and to establish a connection between the presence or absence of Frankia sp. and the alkaloids. In addition, insecticidal activities of the alkaloidal extract were examined. A total of 24 alkaloids were identified, of which 12 have a benzylisoquinoline skeleton, 9 were cyclopeptides, 2 isoquinolines, and 1 aporphine. The presence of cyclopeptide alkaloids is associated with Frankia nodules in the plant root. The alkaloid extracts showed insecticidal activity with mortality dose-dependence and LD50 values between 44 to 71 µg/mL.

Cyclic diarylheptanoids as potential signal compounds during actinorhizal symbiosis between Alnus sieboldiana and Frankia.

The nitrogen-fixing actinomycete Frankia coexists with actinorhizal plants via nodules and supplies nitrogen compounds to the plants. Although communication has been suggested to exist through chemical substances in this nodule symbiosis, the details underlying this mechanism remain elusive. The biphenyl-type diarylheptanoids (BP-CDHs), alnusonol, and alnusdione, previously isolated from the actinorhizal plant A. sieboldiana branch wood, are secondary metabolites that accumulate in a limited number of plant species. However, since relatively widely distributed in actinorhizal plants, we investigated whether adding A. sieboldiana root extracts and these BP-CDHs could affect plant seedlings inoculated with Frankia. The results showed that the addition of root extract or alnusonol significantly increased the number of nodules and lobes more than two times compared with that upon Frankia supplementation only. We also proved that the extracted components of this plant affected nodule symbiosis. Finally, we confirmed through LC-MS that the root extract component contained BP-CDH, alnusonol. The above-described results indicate that BP-CDHs, at leaset alnusonol, might function as signal compounds from the plant side of the actinorhizal symbiosis between A. sieboldiana and Frankia.

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.

Detoxification and reduction of selenite to elemental red selenium by Frankia.

Four Frankia strains (EuI1c, CN3, ACN14a and CcI3) were tested for selenite tolerance. Frankia inefficax strain EuI1c was resistant to selenite with a MIC value of 518.8 µg ml-1. After 48 h incubation with selenite, a reddish precipitate began to appear in these cultures. The red color suggests the reduction of the toxic, soluble, and colorless sodium selenite (Na2SeO32-) to the nontoxic, insoluble, and red colored elemental selenium (Seº). Analysis showed F. inefficax strain EuI1c cultures exposed to 17.3 and 86.5 µg ml-1selenite completely reduced all of the selenite after 5 and 8 days, respectively. When observed under Scanning Electron Microscopy, selenite-resistant F. inefficax strain EuI1c grown with selenite formed nanosphere particles on the hyphal surface as free deposits or in aggregates and inside the hyphae. EDAX analysis of the nanosphere particles determined that they are composed of selenium with up to 27.3-fold increase in intensity as compared to control cells. FTIR Spectroscopy of selenite-stressed cells showed cell surface changes in fatty acids, polysaccharides, carbohydrates and phosphate groups. This result suggests a mechanism for selenite reduction and nanosphere transport through cell membrane in this strain. Native gel electrophoresis of extracted cell-free protein revealed one band showing activity after staining with selenite and NADH. SDS-PAGE analysis revealed the presence of several bands with one dominant band of 37.8 kDa. Mass spectrometry analysis of the bands determined that the main proteins were a periplasmic-binding protein, sulfate ABC transporter and extracellular ligand-binding receptor.

Transcription factors network in root endosymbiosis establishment and development.

Root endosymbioses are mutualistic interactions between plants and the soil microorganisms (Fungus, Frankia or Rhizobium) that lead to the formation of nitrogen-fixing root nodules and/or arbuscular mycorrhiza. These interactions enable many species to survive in different marginal lands to overcome the nitrogen-and/or phosphorus deficient environment and can potentially reduce the chemical fertilizers used in agriculture which gives them an economic, social and environmental importance. The formation and the development of these structures require the mediation of specific gene products among which the transcription factors play a key role. Three of these transcription factors, viz., CYCLOPS, NSP1 and NSP2 are well conserved between actinorhizal, legume, non-legume and mycorrhizal symbioses. They interact with DELLA proteins to induce the expression of NIN in nitrogen fixing symbiosis or RAM1 in mycorrhizal symbiosis. Recently, the small non coding RNA including micro RNAs (miRNAs) have emerged as major regulators of root endosymbioses. Among them, miRNA171 targets NSP2, a TF conserved in actinorhizal, legume, non-legume and mycorrhizal symbioses. This review will also focus on the recent advances carried out on the biological function of others transcription factors during the root pre-infection/pre-contact, infection or colonization. Their role in nodule formation and AM development will also be described.

Antioxidative ability and membrane integrity in salt-induced responses of Casuarina glauca Sieber ex Spreng. in symbiosis with N2-fixing Frankia Thr or supplemented with mineral nitrogen.

The actinorhizal tree Casuarina glauca tolerates extreme environmental conditions, such as high salinity. This species is also able to establish a root-nodule symbiosis with N2-fixing bacteria of the genus Frankia. Recent studies have shown that C. glauca tolerance to high salt concentrations is innate and linked to photosynthetic adjustments. In this study we have examined the impact of increasing NaCl concentrations (200, 400 and 600mM) on membrane integrity as well as on the control of oxidative stress in branchlets of symbiotic (NOD+) and non-symbiotic (KNO3+) C. glauca. Membrane selectivity was maintained in both plant groups at 200mM NaCl, accompanied by an increase in the activity of antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase). Regarding cellular membrane lipid composition, linolenic acid (C18:3) showed a significant decline at 200mM NaCl in both NOD+ and KNO3+ plants. In addition, total fatty acids (TFA) and C18:2 also decreased in NOD+ plants at this salt concentration, resulting in malondialdehyde (MDA) production. Such initial impact at 200mM NaCl is probably due to the fact that NOD+ plants are subjected to a double stress, i.e., salinity and low nitrogen availability. At 400mM NaCl a strong reduction of TFA and C18:3 levels was observed in both plant groups. This was accompanied by a decrease in the unsaturation degree of membrane lipids in NOD+. However, in both NOD+ and KNO3+ lipid modifications were not reflected by membrane leakage at 200 or 400mM, suggesting acclimation mechanisms at the membrane level. The fact that membrane selectivity was impaired only at 600mM NaCl in both groups of plants points to a high tolerance of C. glauca to salt stress independently of the symbiotic relation with Frankia.

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.

Regulation of nitrogen metabolism in salt tolerant and salt sensitive Frankia strains.

Effect of salinity (0, 50, 100, 250, 500 and 750 mM NaCI) was observed on some important physiological parameters of nitrogen metabolism such as nitrate uptake, intracellular and extracellular ammonium status and activities of nitrogenase, nitrate reductase, nitrite reductase and glutamine synthetase among Frankia strains differing in their salt tolerance capacity. Nitrogenase activity closely followed the growth pattern with regular decline on NaCI supplementation. All the other enzymes showed optimum activity at 100 mM and declined further. Co-regulation of the nitrate uptake system and sequential enzyme activities plays a crucial role in governing the nitrogen status of strains during salt stress. HsIil0 experiencing minimum decline in enzyme activities and best possible nitrogen regulation under NaC1 replete condition showed adequate nutritional management. Among all the strains, HsIil0 proved to be salt tolerant on account of above features while the salt sensitive strain HsIi8 lacked the ability to regulate various steps of nitrogen metabolism during salinity, and thus Frankia strain HsIil0 can potentially serve as a potential biofertilizer in the saline soil.

Influence of iron and chelator on siderophore production in Frankia strains nodulating Hippophae salicifolia D. Don.

Effect of iron and chelator on the growth and siderophore production in the ten newly Frankia strains isolated from the root nodules of Hippophae salicifolia D. Don and the two reference strains were studied. Growth of the strains was greatly affected when grown in the iron and EDTA deprived conditions. All the strains were capable of producing both the hydroxamate and catecholate type siderophore that was detected using the Csaky and Arnow assays. Production of siderophore was enhanced in the EDTA replenish condition in contrast to the iron supplemented medium suggesting that EDTA reduces the availability of other free metals and hence creates the stress condition for which the secretion of siderophore is enhanced. A decrease in siderophore production was observed with an increase in iron concentration. Strains HsIi2 and HsIi10 were found to be producing more siderophore than the other strains.

Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca.

cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a beta-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.

Effects of phosphorus and nitrogen on nodulation are seen already at the stage of early cortical cell divisions in Alnus incana.

BACKGROUND AND AIMS: The present work aimed to study early stages of nodulation in a chronological sequence and to study phosphorus and nitrogen effects on early stages of nodulation in Alnus incana infected by Frankia. A method was developed to quantify early nodulation stages in intact root systems in the root hair-infected actinorhizal plant A. incana. Plant tissue responses were followed every 2 d until 14 d after inoculation. Cortical cell divisions were already seen 2 d after inoculation with Frankia. Cortical cell division areas, prenodules, nodule primordia and emerging nodules were quantified as host responses to infection. METHODS: Seedlings were grown in pouches and received different levels of phosphorus and nitrogen. Four levels of phosphorus (from 0.03 to 1 mM P) and two levels of nitrogen (0.71 and 6.45 mM N) were used to study P and N effects on these early stages of nodule development. KEY RESULTS: P at a medium concentration (0.1 mM) stimulated cell divisions in the cortex and a number of prenodules, nodule primordia and emerging nodules as compared with higher or lower P levels. A high N level inhibited early cell divisions in the cortex, and this was particularly evident when the length of cell division areas and presence of the nodulation stages were related to root length. CONCLUSIONS: Extended cortical cell division areas were found that have not been previously shown in A. incana. The results show that effects of P and N are already expressed at the stage when the first cortical cell divisions are induced by Frankia.

Synergistic effect of Glomus intraradices and Frankia spp. on the growth and stress recovery of Alnus glutinosa in an alkaline anthropogenic sediment.

The presence of actinorhizas and arbuscular mycorrhizas may reduce plant stresses caused by adverse soil conditions. A greenhouse experiment was conducted using a sediment with a high pH, resulting from the disposal of waste originated at an acetylene and polyvinylchloride factory, in which Black alder (Alnus glutinosa) seedlings were inoculated either with Glomus intraradices BEG163 (originally isolated from the same sediment), Frankia spp. or both symbionts. After a 6-month growth period, plants inoculated with both symbionts had significantly greater leaf area, shoot height and total biomass when compared with the uninoculated control, the Frankia spp. and the G. intraradices treatments alone. In dual inoculated plants the N and P leaf content was significantly increased. A defoliation experiment was performed to evaluate the stress recovery of A. glutinosa and plants inoculated with both symbionts had a faster leaf regrowth and produced greater numbers of leaves. The dual inoculation resulted in greater numbers of and larger root nodules than when inoculated with Frankia spp. alone. The length and NADH diaphorase activity of the extraradical mycelium of G. intraradices was also significantly greater when dual inoculation was performed. The inoculation with Frankia spp. alone was shown to improve A. glutinosa growth, whereas G. intraradices alone had no positive effect under these environmental conditions. However, when the two symbionts were inoculated together a synergistic effect was observed resulting in a greater benefit for the plants and for both symbionts. The relevance of these findings for the phytorestoration of anthropogenic stressed sediments with high pH is discussed.

Casuarina cunninghamiana tissue extracts stimulate the growth of Frankia and differentially alter the growth of other soil microorganisms.

Aqueous extracts of host plant Casuarina cunninghamiana tissue altered the in vitro growth of its diazotrophic microsymbiont Frankia and a selection of other soil microorganisms. The growth of actinomycetous Frankia strains, 55005. AvcI1, CesI5, CjI82 001, and Cj was stimulated by aqueous extracts of C. cunninghamiana tissue. Green cladodes (photosynthetic branches), unsuberized roots, and suberized roots were more stimulatory than dry cladodes and seed tissue. Aqueous extracts of green cladodes of C. cunninghamiana most stimulated the growth of Casuarina-derived Frankia strains CjI82 001 and 55005. The growth of isolates of soil bacteria Bradyrhizobium japonicum, Arthrobacter globiformis and Bacillus subtillis and of the soil fungi Penicillium oxalicum and Arthroderma cookiellum was either inhibited or not affected by cladode extracts. Cladode extracts stimulated the growth of the actinomycete Streptomyces albus and the fungus Rhizopus homothallicus. The magnitude (as great as 100%) of the increase in growth caused by tissue extracts for the Casuarina-derived Frankia strains relative to other soil microbes suggests a host-specific enhancement of the microsymbiont.

Coordinate increase in activity of proteinase subunits of the 1300-kDa megaproteinase of Frankia strain BR: role of carbon source depletion and extracellular metabolites.

We have recently described the presence of a high molecular mass multicatalytic proteinase complex (megaproteinase; 28 S, 1300 kDa) in Frankia strain BR. The complex dissociates into 11 low molecular mass proteinase subunits (40-19 kDa) when subjected to sodium dodecyl sulfate - gelatin - polyacrylamide gel electrophoresis. We show here that the activity of these proteinase subunits strongly increased after cessation of growth in stirred BAP-PCM mineral medium. Subsequent addition of either BAP medium components or sodium propionate alone, as carbon source, to a Frankia culture at the end of the exponential growth phase was found to prolong growth for 1 additional day, and to delay the increase in activity of the proteinase subunits for 3 days after cessation of growth. Addition of ammonium chloride alone, as nitrogen source, had no effect. On the other hand, when Frankia cells in the late exponential phase (3 days) were resuspended in a culture filtrate recovered from a 5-day-old culture and supplemented with BAP-PCM medium components, the biomass yield decreased to about 50%. Also, the activity of the proteinase subunits increased as soon as growth ceased. The ability of this culture filtrate to inhibit growth and stimulate the activity of proteinase subunits was partially lost by heating or was largely removed by DEAE-cellulose treatment. Thus, our findings indicate an extracellular control of Frankia megaproteinase activity, suggesting that carbon source depletion and probably accumulation of heat-sensitive growth-inhibiting metabolites in the medium are determining factors.