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      Volume 38, Issue 4

      November 2013,   pages  675-823

    • Editorial

      Arvind K Misra

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    • Introduction: A brief history of Frankia and actinorhizal plants meetings

      Philippe Normand

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    • Micromonospora is a normal occupant of actinorhizal nodules

      Lorena Carro Petar Pujic Martha E Trujillo Phillipe Normand

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      Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing.

      Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis.

      Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.

    • First report on the occurrence of the uncultivated cluster 2 Frankia microsymbionts in soil outside the native actinorhizal host range area

      Imen Nouioui Imed Sbissi Faten Ghodhbane-Gtari Kawtar Fikri Benbrahim Philippe Normand Maher Gtari

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      The occurrence of uncultivated Frankia was evaluated in Tunisian soils by a plant-trapping assay using Coriaria myrtifolia seedlings. Despite the lack of this compatible host plant for more than two centuries, soil-borne Frankia cells were detected in one sampled soil as shown by the development of root nodules on 2-year-old seedlings. Based on glnA sequences, Tunisian trapped Frankia strains belong to the uncultivated cluster 2 strains that associate with other Coriaria species and also with Ceanothus, Datisca and Rosaceae actinorhizal species. This is the first report on the occurrence of Frankia cluster 2 strains in soils from areas lacking compatible host plant groups.

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

      Rediet Oshone Samira R Mansour Louis S Tisa

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      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.

    • Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

      K H Richau R L Kudahettige P Pujic N P Kudahettige A Sellstedt

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      The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).

    • Codon-optimized antibiotic resistance gene improves efficiency of transient transformation in Frankia

      Ken-ichi Kucho Kentaro Kakoi Masatoshi Yamaura Mari Iwashita Mikiko Abe Toshiki Uchiumi

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      Frankia is a unique actinobacterium having abilities to fix atmospheric dinitrogen and to establish endosymbiosis with trees, but molecular bases underlying these interesting characteristics are poorly understood because of a lack of stable transformation system. Extremely high GC content of Frankia genome (> 70%) can be a hindrance to successful transformation. We generated a synthetic gentamicin resistance gene whose codon usage is optimized to Frankia (fgmR) and evaluated its usefulness as a selection marker using a transient transformation system. Success rate of transient transformation and cell growth in selective culture were significantly increased by use of fgmR instead of a native gentamicin resistance gene, suggesting that codon optimization improved translation efficiency of the marker gene and increased antibiotic resistance. Our result shows that similarity in codon usage pattern is an important factor to be taken into account when exogenous transgenes are expressed in Frankia cells.

    • What stories can the Frankia genomes start to tell us?

      Louis Tisa Nicholas Beauchemin Maher Gtari Arnab Sen Luis G Wall

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      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.

    • Characterization of pseudogenes in members of the order Frankineae

      Saubashya Sur Sangita Saha Louis S Tisa Asim K Bothra Arnab Sen

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      Pseudogenes are defined as non-functional relatives of genes whose protein-coding abilities are lost and are no longer expressed within cells. They are an outcome of accumulation of mutations within a gene whose end product is not essential for survival. Proper investigation of the procedure of pseudogenization is relevant for estimating occurrence of duplications in genomes. Frankineae houses an interesting group of microorganisms, carving a niche in the microbial world. This study was undertaken with the objective of determining the abundance of pseudogenes, understanding strength of purifying selection, investigating evidence of pseudogene expression, and analysing their molecular nature, their origin, evolution and deterioration patterns amongst domain families. Investigation revealed the occurrence of 956 core pFAM families sharing common characteristics indicating co-evolution. WD40, Rve_3, DDE_Tnp_IS240 and phage integrase core domains are larger families, having more pseudogenes, signifying a probability of harmful foreign genes being disabled within transposable elements. High selective pressure depicted that gene families rapidly duplicating and evolving undoubtedly facilitated creation of a number of pseudogenes in Frankineae. Codon usage analysis between protein-coding genes and pseudogenes indicated a wide degree of variation with respect to different factors. Moreover, the majority of pseudogenes were under the effect of purifying selection. Frankineae pseudogenes were under stronger selective constraints, indicating that they were functional for a very long time and became pseudogenes abruptly. The origin and deterioration of pseudogenes has been attributed to selection and mutational pressure acting upon sequences for adapting to stressed soil environments.

    • Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family

      Subarna Thakur Asim K Bothra Arnab Sen

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      Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD&K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity.We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication.

    • Growth response of Casuarina equisetifolia Forst. rooted stem cuttings to Frankia in nursery and field conditions

      A Karthikeyan K Chandrasekaran M Geetha R Kalaiselvi

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      Casuarina equisetifolia Forst. is a tree crop that provides fuel wood, land reclamation, dune stabilization, and scaffolding for construction, shelter belts, and pulp and paper production. C. equisetifolia fixes atmospheric nitrogen through a symbiotic relationship with Frankia, a soil bacterium of the actinobacteria group. The roots of C. equisetifolia produce root nodules where the bacteria fix atmospheric nitrogen, which is an essential nutrient for all plant metabolic activities. However, rooted stem cuttings of elite clones of C. equisetifolia by vegetative propagation is being planted by the farmers of Pondicherry as costeffective method. As the vegetative propagation method uses inert material (vermiculite) for rooting there is no chance for Frankia association. Therefore after planting of these stocks the farmers are applying 150 kg of di-ammonium phosphate (DAP)/acre/year. To overcome this fertilizer usage, the Frankia-inoculated rooted stem cuttings were propagated under nursery conditions and transplanted in the nutrient-deficient soils of Karaikal, Pondicherry (India), in this study. Under nursery experiments the growth and biomass of C. equisetifolia rooted stem cuttings inoculated with Frankia showed 3 times higher growth and biomass than uninoculated control. These stocks were transplanted and monitored for their growth and survival for 1 year in the nutrient-deficient farm land. The results showed that the rooted stem cuttings of C. equisetifolia significantly improved growth in height (8.8 m), stem girth (9.6 cm) and tissue nitrogen content (3.3 mg g−1) than uninoculated controls. The soil nutrient status was also improved due to inoculation of Frankia.

    • Indigenous actinorhizal plants of Australia

      Nishath K Ganguli Ivan R Kennedy

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      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.

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

      Hiroyuki Tobita Shigeaki F Hasegawa Kenichi Yazaki Masabumi Komatsu Mitsutoshi Kitao

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      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.

    • Characterization of haemoglobin from Actinorhizal plants -- An in silico approach

      Sanghati Bhattacharya Arnab Sen Subarna Thakur Louis S Tisa

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      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.

    • Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings

      Mhathung Yanthan Arvind K Misran

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      Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

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

      Arvind K Goyal Tanmayee Mishra Malay Bhattacharya Pallab Kar Arnab Sen

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      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.

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

      Sushil Kumar Middha Arvind Kumar Goyal Syed Faizan Nethramurthy Sanghmitra Bharat Chandra Asistha Talambedu Usha

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      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.

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

      Chonglu Zhong Samira Mansour Mathish Nambiar-Veetil Didier Bogusz Claudine Franche

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      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.

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