Plant Growth-Promoting Active Metabolites from Frankia spp. of Actinorhizal Casuarina spp.

In agriculture, plant growth enrichment via plant growth stimulating microbes has been recognized as an emergency, it is used as an alternatives to chemical pesticides and growth stimulants. The phytopathogens cause various diseases such as blister bark; stem cankers, and pink and brown rot diseases besides affect the growth frequency of Casuarina spp. toward biotic and abiotic stresses. Bio-control and plant growth-promoting potential of native Frankia isolates from Casuarina spp. in Tamil Nadu, India, was not much explored. Hence, in the present study, we are investigating the plant growth improvement activity and phytopathogen control in Casuarina spp. The Frankia sp. DDNSF-01 and Frankia casuarinae DDNSF-02 were isolated and identified from the root nodules of Casuarina spp. Additionally, it is recognized for plant growth promoter activity and in vitro antimicrobial activity against phytopathogens including Pseudomonas sp. and Colletotrichum sp. The plant growth regulators including IAA, siderophore, ammonia production, and phosphate solubilization were found out. Therefore, the formation of the most significant plant growth-promoting phytohormone IAA was confirmed by UV, FT-IR, TLC, HPLC, HPTLC, and NMR spectrum. Bioactive metabolites including methyl 4-hydroxybenzoate, dodecanoic acid, and some novel flavonoids were identified. Therefore, various growth regulators such as L-aspartic acid, 1H-indole-3-carboxaldehyde were confirmed by GC-MS spectra. The present findings conclude Frankia spp. as efficient plant growth enhancement mediator and also inhibit the phytopathogens.

The PEG-responding desiccome of the alder microsymbiont Frankia alni.

Actinorhizal plants are ecologically and economically important. Symbiosis with nitrogen-fixing bacteria allows these woody dicotyledonous plants to colonise soils under nitrogen deficiency, water-stress or other extreme conditions. However, proteins involved in xerotolerance of symbiotic microorganisms have yet to be identified. Here we characterise the polyethylene glycol (PEG)-responding desiccome from the most geographically widespread Gram-positive nitrogen-fixing plant symbiont, Frankia alni, by next-generation proteomics, taking advantage of a Q-Exactive HF tandem mass spectrometer equipped with an ultra-high-field Orbitrap analyser. A total of 2,052 proteins were detected and quantified. Under osmotic stress, PEG-grown F. alni cells increased the abundance of envelope-associated proteins like ABC transporters, mechano-sensitive ion channels and Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated (cas) components. Conjointly, dispensable pathways, like nitrogen fixation, aerobic respiration and homologous recombination, were markedly down-regulated. Molecular modelling and docking simulations suggested that the PEG is acting on Frankia partly by filling the inner part of an up-regulated osmotic-stress large conductance mechanosensitive channel.

Salt stress-induced changes in antioxidative defense system and proteome profiles of salt-tolerant and sensitive Frankia strains.

An appreciation of comparative microbial survival is most easily done while evaluating their adaptive strategies during stress. In the present experiment, antioxidative and whole cell proteome variations based on spectrophotometric analysis and SDS-PAGE and 2-dimensional gel electrophoresis have been analysed among salt-tolerant and salt-sensitive Frankia strains. This is the first report of proteomic basis underlying salt tolerance in these newly isolated Frankia strains from Hippophae salicifolia D. Don. Salt-tolerant strain HsIi10 shows higher increment in the contents of superoxide dismutase, catalase and ascorbate peroxidase as compared to salt-sensitive strain HsIi8. Differential 2-DGE profile has revealed differential profiles for salt-tolerant and salt-sensitive strains. Proteomic confirmation of salt tolerance in the strains with inbuilt efficiency of thriving in nitrogen-deficient locales is a definite advantage for these microbes. This would be equally beneficial for improvement of soil nitrogen status. Efficient protein regulation in HsIi10 suggests further exploration for its potential use as biofertilizer in saline soils.

Isolation and analysis of vitamin B12 from plant samples.

Based on increased demands of strict vegetarians, an investigation of vitamin B12 content in plant sources, was carried out. The vitamin B12 concentration was determined by RP-HPLC with UV detection, after prior matrix isolation by immunoaffinity chromatography (IAC). Vitamin B12 was extracted in the presence of sodium cyanide, to transform all forms of cobalamin into cyanocobalamin. Diode array detector was used to monitor vitamin B12, after its chromatographic separation under gradient elution with a mobile phase consisting of acetonitrile and trifluoroacetic acid 0.025% (w/v). The method demonstrated excellent linearity with a limit of detection 0.004µg/ml. The method precision was evaluated for plant samples and it was below 0.7% (n=6). Significant amounts of vitamin B12 in plants were detected in Hippophae rhamnoides (37µg/100g dry weight), in Elymus (26µg/100g dry weight) and in Inula helenium (11µg/100g dry weight).

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3.

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.

Typing of nitrogen-fixing Frankia strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry.

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was evaluated as a technique to characterize strains of the nitrogen-fixing actinomycete Frankia. MALDI-TOF MS reliably distinguished 37 isolates within the genus Frankia and assigned them to their respective host infection groups, i.e., the Alnus/Casuarina and the Elaeagnus host infection groups. The assignment of individual strains to sub-groups within the respective host infection groups was consistent with classification based on comparative sequence analysis of nifH gene fragments, confirming the usefulness of MALDI-TOF MS as a rapid and reliable tool for the characterization of Frankia strains.

Diversity of Frankia strains nodulating Hippophae salicifolia D. Don using FAME profiling as chemotaxonomic markers.

Twelve Frankia strains isolated from Hippophae salicifolia D. Don or Alnus glutinosa or Comptonia peregrine, showed the significant variation in fatty acid composition viz. palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidic acid (20:0) and erucic acid (22:1) suggesting the strain specific variability among the Frankia strains. Presence of Erucic acid (22:1), a major component of the oil obtained from the seeds of Brassica sp., albeit in lesser amount in the few studied frankial strains, is the first report. Cluster analysis on the basis of fatty acid composition suggests the presence of two distinct clusters with similitude coefficient ranging from 0.75 to 1.00. Cluster I with HsIi2 showed great divergence from other 11 frankial strains (Cluster II). The two sub groups were distinguished in cluster II: IIa contained five strains isolated from H. salicifolia and these strains are distantly related to the strains of cluster IIb isolated from different host. There is high degree of similarity among the frankial strains of Cluster IIb which suggests that the frankial strains might be evolved from the same ancestor. FAME profiling might be useful tool in the study of polyphasic approach based taxonomy and phylogenetic relationship.

Truncated hemoglobins in Frankia CcI3: effects of nitrogen source, oxygen concentration, and nitric oxide.

Frankia strain CcI3 produces 2 truncated hemoglobins, HbN and HbO. Using ion-exchange chromatography, we characterized the expression of the relative amounts of HbN and HbO in -N (nitrogen-fixing) cultures and +N (nitrogen-supplemented) cultures. The -N cultures maintained an approximately constant ratio of HbO to HbN throughout the life of the culture, with HbO constituting 80%-85% of the total hemoglobin produced. In contrast, in +N cultures, HbN was observed to increase over time and HbO decreased. Total hemoglobin as a fraction of total protein was approximately constant throughout the growth phase in -N cultures, while it decreased somewhat in +N cultures. Subjecting -N cultures to a NO generator resulted in increased production of HbN, relative to the controls. Nitrite accumulated in +N cultures, but not in -N cultures. This suggests that the greater amount of HbN in +N cultures might be due to NO produced by the reduction of nitrite. The effects of O2 concentration were determined in +N cultures. Cultures grown in 1% O2 produced about 4 times more HbO than cultures grown in 20% O2. Overall, these results provide evidence for a role of HbN in NO oxidation and for a role of HbO in adaptation to low oxygen concentrations.

Diminished exoproteome of Frankia spp. in culture and symbiosis.

Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N(2) fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

El efecto de la interacción Frankia - micorrizas - micronutrientes en el establecimiento de árboles Aliso (Alnus acuminata) en sistemas silvopastoriles / The effects of the interaction Frankia - mycorrhizal fungi - microelements in the establishment of Alder trees (Alnus acuminata) in tree grass systems

Frankia es un género de microorganismos llamados actinomicetos capaces de inducir la formación de nódulos radiculares fijadores de nitrógeno atmosférico en algunas angiospermas no leguminosas, denominadas plantas actinorrízicas. El desarrollo de fijación de Nitrógeno (N) simbiótico envuelve múltiples procesos que se llevan a cabo en compartimentos subcelulares endosimbióticos donde Francia produce los nódulos de la raíz que convierten el N2 en forma combinada. La simbiosis micorriza y nódulo es generalmente sinérgica, promueve el crecimiento vegetal, la multiplicación de las micorrizaspropicia un ambiente de mayor competencia, no favorable para los agentes patógenos. A lo anterior se debe sumar la participación de microelementos como el molibdeno(Mo), el cual es constituyente dela nitrogenasa y cuya deficiencia en el medio causa un efecto directo y negativo en la fijación del N; con el boro(B,) donde su deficiencia produce una desestabilización de la cubierta protectora contra el oxígeno, afectando considerablemente la nodulación de la planta y con el cobalto(Co), forman un componente estructural de la coenzima de la vitamina B12 en el proceso de fijación de N. Sin embargo, muchos aspectos relacionados de estos microelementos en la interrelación y de la simbiosis micorriza y Frankia se desconocen. Por lo anterior, es importante determinar sus efectos sobre los microorganismos del suelo y el desarrollo de los nódulos; para intepretar una óptima manifestación, sin embargo su complejidad hace que su efecto no sea predecible bajo todas las condiciones ni para todas las especies. Por lo tanto, es importante incentivar la investigación al respecto, así como profundizar en el conocimiento de sus principios de funcionamiento, interacciones y mostrar los resultados encontradosen su uso en árboles promisiorios para sistemas silvopastoriles trópico alto como el aliso (Agnus acuminata), especie forestal útil en la solución de problemas de degradación de suelos y deforestación.

A hydrogen-evolving enzyme is present in Frankia sp. R43.

The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen,which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.

Why boron?

It is now more than 80 years since boron was convincingly demonstrated to be essential for normal growth of higher plants. However, its biochemical role is not well understood at the moment. Several recent reviews propose that B is implicated in three main processes: keeping cell wall structure, maintaining membrane function, and supporting metabolic activities. However, in the absence of conclusive evidence, the primary role of boron in plants remains elusive. Besides plants, growth of specific bacteria, such as heterocystous cyanobacteria and the recently reported actinomycetes of the genus Frankia, requires B, particularly for the stability of the envelopes that control the access of the nitrogenase-poisoning oxygen when they grow under N2-fixing conditions. Likewise, a role for B for animal embryogenesis and other developmental processes is being established. Finally, a new feature of the role of boron comes from signaling mechanisms for communication among bacteria and among legumes and rhizobia leading to N2-fixing symbiosis, and it is possible that new roles for B, based on its special chemistry and its interaction with Ca would appear in the world of signal transduction pathways. In conclusion, the diversity of roles played by B might indicate that either the micronutrient is involved in numerous processes or that its deficiency has a pleiotropic effect. The arising question is why such an element? Since all of the roles clearly established for B are related to its capacity to form diester bridges between cis-hydroxyl-containing molecules, we propose that the main reason for B essentiality is the stabilization of molecules with cis-diol groups turning them effective, irrespectively of their function.