Casuarina glauca: A model tree for basic research in actinorhizal symbiosis.

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.

In silico–based combinatorial pharmacophore modelling and docking studies of GSK-3β and GK inhibitors of Hippophae.

Type 2 diabetes is an inevitably progressive disease, with irreversible β cell failure. Glycogen synthase kinase and Glukokinase, two important enzymes with diverse biological actions in carbohydrate metabolism, are promising targets for developing novel antidiabetic drugs. A combinatorial structure-based molecular docking and pharmacophore modelling study was performed with the compounds of Hippophae salicifolia and H. rhamnoides as inhibitors. Docking with Discovery Studio 3.5 revealed that two compounds from H. salicifolia, viz Lutein D and an analogue of Zeaxanthin, and two compounds from H. rhamnoides, viz Isorhamnetin-3-rhamnoside and Isorhamnetin-7-glucoside, bind significantly to the GSK-3 β receptor and play a role in its inhibition; whereas in the case of Glucokinase, only one compound from both the plants, i.e. vitamin C, had good binding characteristics capable of activation. The results help to understand the type of interactions that occur between the ligands and the receptors. Toxicity predictions revealed that none of the compounds had hepatotoxic effects and had good absorption as well as solubility characteristics. The compounds did not possess plasma protein-binding, crossing blood–brain barrier ability. Further, in vivo and in vitro studies need to be performed to prove that these compounds can be used effectively as antidiabetic drugs.

Evaluation of phytochemical constituents and antioxidant activity of selected actinorhizal fruits growing in the forests of Northeast India.

Hippophae salicifolia, Elaeagnus pyriformis, Myrica esculenta and M. nagi are actinorhizal plants growing in the sacred forests of Northeast India with multipurpose uses. The present investigation was undertaken to determine the phenol, flavonoid and flavonol contents of the fresh fruit juice of these plant species including the antioxidant potential by means of DPPH, H2O2 and NO scavenging activity and FRP. The total phenolic, flavonoid and flavonol contents of fruit juice ranged from 321.68±0.06 to 76.67±0.01 mg/g GAE, 272.92±0.07 to 20.12±0.02 mg/g QE and 258.92±0.08 to 18.72±0.02 mg/g QE, respectively. At 2.0 mg/mL concentration, DPPH scavenging activity was found to be the highest in M. esculenta (89.62%) and the lowest in E. pyriformis (17.58%). The reducing power activity was found significantly higher in H. salicifolia juice, which increased with increase in concentration. The H2O2 scavenging activity of H. salicifolia juice was found to be as high as 98.78%, while Elaeagnus juice was found to be less effective with just 48.90%. Juice of H. salicifolia showed the greatest NO scavenging effect of 75.24% as compared to juice of E. pyriformis, where only 37.54% scavenging was observed at the same concentration. Taking into account all the experimental data, it can be said that the fruits of H. salicifolia and both M. nagi and M. esculenta have good antioxidant activity compared to fruits of E. pyriformis.

Characterization of haemoglobin from Actinorhizal plants – An in silico approach.

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.

Growth and N2 fixation in an Alnus hirsuta (Turcz.) var. sibirica stand in Japan.

To estimate the N2 fixation ability of the alder (Alnus hirsuta (Turcz.) var. sibirica), we examined the seasonal variation in nitrogenase activity of nodules using the acetylene reduction method in an 18-year-old stand naturally regenerated after disturbance by road construction in Japan. To evaluate the contribution of N2 fixation to the nitrogen (N) economy in this alder stand, we also measured the phenology of the alder, the litterfall, the decomposition rate of the leaf litter, and N accumulation in the soil. The acetylene reduction activity per unit nodule mass (ARA) under field conditions appeared after bud break, peaked the maximum in midsummer after full expansion of the leaves, and disappeared after all leaves had fallen. There was no consistent correlation between ARA and tree size (dbh). The amount of N2 fixed in this alder stand was estimated at 56.4 kg ha−1 year−1 when a theoretical molar ratio of 3 was used to convert the amount of reduced acetylene to the amount of fixed N2. This amount of N2 fixation corresponded to the 66.4% of N in the leaf litter produced in a year. These results suggested that N2 fixation still contributed to the large portion of N economy in this alder stand.

Indigenous actinorhizal plants of Australia.

Indigenous species of actinorhizal plants of Casuarinaceae, Elaeagnaceae and Rhamnaceae are found in specific regions of Australia. Most of these plants belong to Casuarinaceae, the dominant actinorhizal family in Australia. Many of them have significant environmental and economical value. The other two families with their indigenous actinorhizal plants have only a minor presence in Australia. Most Australian actinorhizal plants have their native range only in Australia, whereas two of these plants are also found indigenously elsewhere. The nitrogen-fixing ability of these plants varies between species. This ability needs to be investigated in some of these plants. Casuarinas form a distinctive but declining part of the Australian landscape. Their potential has rarely been applied in forestry in Australia despite their well-known uses, which are being judiciously exploited elsewhere. To remedy this oversight, a programme has been proposed for increasing and improving casuarinas that would aid in greening more regions of Australia, increasing the soil fertility and the area of wild life habitat (including endangered species). Whether these improved clones would be productive with local strains of Frankia or they need an external inoculum of Frankia should be determined and the influence of mycorrhizal fungi on these clones also should be investigated.

What stories can the Frankia genomes start to tell us.

Among the Actinobacteria, the genus Frankia is well known for its facultative lifestyle as a plant symbiont of dicotyledonous plants and as a free-living soil dweller. Frankia sp. strains are generally classified into one of four major phylogenetic groups that have distinctive plant host ranges. Our understanding of these bacteria has been greatly facilitated by the availability of the first three complete genome sequences, which suggested a correlation between genome size and plant host range. Since that first report, eight more Frankia genomes have been sequenced. Representatives from all four lineages have been sequenced to provide vital baseline information for genomic approaches toward understanding these novel bacteria. An overview of the Frankia genomes will be presented to stimulate discussion on the potential of these organisms and a greater understanding of their physiology and evolution.

Effect of salt stress on the physiology of Frankia sp strain CcI6.

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.

Caracterização morfológica, fisiológica e infectividade em planta de estirpes de Frankia isoladas de nódulos de Casuarina / Morphological, physiological and plant infectivity characterization of Frankia strains isolated from Casuarina’s nodules

Frankia são microrganismos capazes de fixar N2 quando em simbiose com espécies arbóreas, denominadas plantas actinorrízicas. Este estudo foi conduzido para caracterizar morfologicamente, fisiologicamente e avaliar a nodulação de quatro estirpes referência de Frankia (HFPCcI3, JCT287, KB5 e F59) e 12 estirpes(IPRF) isoladas de nódulos de Casuarina. Todas as estirpes (Referência e IPRF) foram Gram-positivas e 50% foram “acid-fast”. As estirpes de Frankia alcalinizaram o meio de cultura, exceto IPRF006, IPRF008e IPRF010. As colônias das estirpes F59, IPRF002, IPRF004, IPRF005 e IPRF011, produziram melanina. Entre as estirpes referência, apenas a JCT287 cresceu no meio de cultura com pH 5,5, enquanto no meio com pH 6,0 as duas, JCT287 e KB5, apresentaram crescimento. A análise de regressão mostrou uma relação linear (Y = 67,56+ 3,88X e R2=0,5862, p<0,05) e coeficiente de correlação de Pearson (r=0,766,p<0,05) entre número de esporos e hifas (120 a 480 UFC mL-1) e proteína total (18 a 145 µg mL-1). Foi observado que a estirpe F59 apresentou uma maior proporção de proteína total (50%) do que a JCT287(7,0%) e, que as estirpes IPRF mostraram valores entre 17,5 e 29,3%. Todas as estirpes produziram compostos indólicos com valores variando de 5,9 a 98,8 µM.

Frankia are soil microorganisms that form symbiosis with roots of tree species called actinorhizal plants and are capable of fixing atmospheric N2. This study was carried out to characterize morphologically, physiologically and to assess the nodulation of four Frankia reference strains (HFPCcI3, JCT287, KB5and F59) and 12 (IPRF) isolated from root nodules of Casuarina plants. All strains (Reference and IPRF)were characterized as Gram-positive and 50% as acid-fast. The Frankia strains produced alkali in the culture medium, except the IPRF006, IPRF008 and IPRF010. The colonies of strains F59, IPRF002, IPRF004,IPRF005, and IPRF011 produced melanin. Among reference strains, only JCT287 grew in culture mediawith pH 5.5, while with pH 6.0 both strains JCT287 and KB5 presented growth. The regression analysis showed a linear relationship (Y = 67.56+ 3.88X and R2=0.5862, p<0.05) and Pearson’s correlation coefficient(r=0.766, p<0.05) between number of spores and hyphae (120 to 480 UFC mL-1) and total protein (18 to 145µg mL-1). It was observed that the strains F59 had a higher proportion of total protein 50.0% than JCT287with 7.0% and that the IPRF strains showed values between 17.5 and 29.3%. All strains presented ability to produce indolic compounds in growth media with values ranging from 5.9 to 98.8 µM.