Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables.

The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance.

Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania.

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

Evaluation of the Nutraceutical and Cosmeceutical Potential of Two Cultivars of Rubus fruticosus L. under Different Cultivation Conditions.

BACKGROUND: The starting point for the development of new, functional products derived from Rubus fruticosus L. is to determine the optimal cultivation conditions that produce maximal yield of fruits containing desirable bioactive properties. Towards that goal, the effect of soil, soil/peat mixture and light intensity on the nutraceutical and cosmeceutical potential of two cultivars ('Thornfree' and 'Loch Ness') of Rubus fruticosus L. were evaluated. METHODS: The assessment was carried out employing a range of methods for evaluating fruit properties associated with promoting good health such as total antioxidant capacity, secondary metabolites content (vitamin C, polyphenols, flavonoids and anthocyanins) and inhibition analysis of skin-regulating enzymes. RESULTS: 'Thornfree' cultivar produced fruits in all light conditions, while 'Loch Ness' did not produce fruits in low light conditions. The results showed that in Rubus fruticosus L. fruit, the chemical composition and bioactivity are strongly affected by both genetics factors and growing conditions. Extract from 'Thornfree' fruits obtained under low light and soil/peat conditions displayed superior properties such as high antioxidant capacity, high concentrations of phenolics, flavonoids and anthocyanins and high inhibitory potency towards the enzymes tyrosinase and elastase. This extract was used for the development of a topical skin care cream with excellent compatibility and stability. CONCLUSION: Our findings conclude that Rubus fruticosus L. cultivation may be efficiently and effectively manipulated through conventional cultivation techniques to produce promising bioactive ingredients with potential use in commercial cosmetics and pharmaceuticals.

Functional and Catalytic Characterization of the Detoxifying Enzyme Haloalkane Dehalogenase from Rhizobium leguminosarum.

BACKGROUND: Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/ß-hydrolases which catalyze the irreversible cleavage of carbon-halogen bonds of haloalkanes, producing an alcohol, a halide and a hydrogen ion. Haloalkanes are acutely toxic to animals and humans and their toxic effects are mainly observed in the liver, kidneys and central nervous system. OBJECTIVE: In the present work, the haloalkane dehalogenase from Rhizobium leguminosarum bv. trifolii (DrlA) was characterized. METHOD: Reverse transcription polymerase chain reaction analysis and enzyme activity assays revealed that the DrlA gene expression in R. leguminosarum bv. trifolii is induced by 1,2- dibromoethane (1,2-DBE) during the early exponential phase. The gene of the enzyme was isolated, cloned and expressed in E. coli Rosetta (DE3). RESULTS: Recombinant DrlA displays its high catalytic activity towards 1,2-DBE and the long-chain haloalkane 1-iodohexane. Limited activity was observed for other aliphatic and cyclic haloalkanes, indicating that the enzyme displays restricted substrate specificity, compared to other bacterial HLDs. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the HLD-II subfamily and shares the same overall fold and domain organization as other bacterial HLDs, however major variations were identified at the hydrophobic substrate-binding cavity, the cap domain and the entrance of the main tunnel that affect the size of the active site pocket and the substrate recognition mechanism. CONCLUSION: This work sheds new light on the environmental fate and toxicity of 1,2-DBE and provides new knowledge on the structure, function and diversity of HLDs for developing applications in toxicology.

Symbiont abundance is more important than pre-infection partner choice in a Rhizobium - legume mutualism.

It is known that the genetic diversity of conspecific rhizobia present in root nodules differs greatly among populations of a legume species, which has led to the suggestion that both dispersal limitation and the local environment affect rhizobial genotypic composition. However, it remains unclear whether rhizobial genotypes residing in root nodules are representative of the entire population of compatible symbiotic rhizobia. Since symbiotic preferences differ among legume populations, the genetic composition of rhizobia found within nodules may reflect the preferences of the local hosts, rather than the full diversity of potential nodulating rhizobia present in the soil. Here, we assessed whether Vicia cracca legume hosts of different provenances select different Rhizobium leguminosarum genotypes than sympatric V. cracca hosts, when presented a natural soil rhizobial population. Through combining V. cracca plants and rhizobia from adjacent and more distant populations, we found that V. cracca hosts are relatively randomly associated with rhizobial genotypes. This indicates that pre-infection partner choice is relatively weak in certain legume hosts when faced with a natural population of rhizobia.

RAPD-inferred genetic variability of some indigenous Rhizobium leguminosarum isolates from red clover (Trifolium pratense L.) nodules.

The application of commercial rhizobial inoculants to legume crops is proving to be an alternative to synthetic fertilizer use. The challenge for sustainable agriculture resides in the compatibility between crop, inoculants and environmental conditions. The evaluation of symbiotic efficiency and genetic diversity of indigenous rhizobial strains could lead to the development of better inoculants and increased crop production. The genetic variability of 32 wild indigenous rhizobial isolates was assessed by RAPD (Random Amplified Polymorphic DNA). The strains were isolated from red clover (Trifolium pratense L.) nodules from two distinct geographical regions of Northern and Eastern Romania. Three decamer primers were used to resolve the phylogenetic relationships between the investigated isolates. Cluster analysis revealed a high diversity; most strains clustered together based on their geographical location.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.

Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells.

One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays.

BACKGROUND: Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species-specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS: When collections of non-steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100-fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION: The present study demonstrated that species-specific ecdysone-agonist-based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification.

Juvenile hormone analogs do not affect directly the activity of the ecdysteroid receptor complex in insect culture cell lines.

During insect development, ecdysteroids and juvenile hormones (JHs) interact to regulate larval growth, metamorphosis and reproduction but the molecular mechanisms by which both hormones influence each other's activity remain unknown. Because of their ease of use and straightforward genetic manipulation, insect cell lines often have been used to clarify the actions and interactions of hormones at the molecular level. Here we report on the use of two insect culture cell lines, Drosophila melanogaster S2 and Bombyx mori Bm5 cells, to investigate two molecular processes in which ecdysteroids and JH have been shown to interact: (1) direct modulation of the activity of the ecdysteroid receptor transcription complex and (2) interference at the level of induction of the primary gene E75. Our data do not support JH analogs (JHAs) acting through the above processes: 'antagonism' of ecdysteroid receptor activity by JHAs correlated with cytotoxicity and induction of E75 expression by JHAs was not demonstrated. However, we confirm previous studies in which it was observed that methoprene can partially reverse the growth inhibition by 20E in S2 cells (but not Bm5 cells). Therefore, the molecular mechanism by which both hormones influence each other's activity to regulate cell growth in S2 cells remains unknown.

Spatial and temporal organization of sucrose metabolism in Lotus japonicus nitrogen-fixing nodules suggests a role for the elusive alkaline/neutral invertase.

Symbiotic nitrogen fixation (SNF) in legume nodules is a highly energy demanding process, fuelled by plant-supplied carbohydrates mainly in the form of sucrose. In this study, we have combined molecular and biochemical approaches in order to study the spatial and temporal organisation of sucrose metabolism in nitrogen-fixing nodules of the model legume Lotus japonicus, with an emphasis on the neglected role of alkaline/neutral invertase. For this purpose, a full-length cDNA clone coding for an alkaline/neutral invertase isoform, termed LjInv1, was identified in a L. japonicus mature nodule cDNA libraries. Alkaline/neutral invertase activity was also found to be the predominant invertase activity in mature nodules. Real-time reverse-transcription polymerase chain reaction analysis was used in order to study the temporal expression patterns of LjInv1 in parallel with genes encoding acid invertase and sucrose synthase (SuSy) isoforms, and enzymes involved in the subsequent hexose partitioning including hexokinase, phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI). The spatial organisation of sucrose metabolism was studied by in situ localisation of LjInv1 transcripts and alkaline/neutral invertase activity, and SuSy protein during nodule development. Furthermore, the spatial organisation of hexose metabolism was investigated by histochemical localisation of hexokinase, PGM and PGI activities in mature nodules. The results considered together indicate that alkaline/neutral invertase could contribute to both the Glc-1-P and Glc-6-P pools in nodules, fuelling both biosynthetic processes and SNF. Furthermore, transcript profiling analysis revealed that genes coding for hexokinase and putative plastidic PGM and PGI isoforms are upregulated during the early stages of nodule development, while the levels of transcripts corresponding to cytosolic PGM and PGI isoforms remained similar to uninfected roots, indicating a possible role of LjInv1 in producing hexoses for starch production and other biosynthetic processes in developing nodules.

Baculovirus-mediated gene delivery into Mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression.

Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

Ornithine decarboxylase and arginine decarboxylase gene transcripts are co-localized in developing tissues of Glycine max etiolated seedlings.

Unlike other eukaryotes, which synthesize polyamines (PA) only from ornithine, plants possess an additional pathway utilizing arginine as a precursor. In this study, we have identified cDNA clones coding for a Glycine max ornithine decarboxylase (ODC, EC 4.1.1.7) and an arginine decarboxylase (ADC, EC 4.1.1.19). Expression analysis using semi-quantitative RT-PCR approach revealed that both genes coding for enzymes involved in putrescine biosynthesis (ODC and ADC) were found in most plant organs examined. Significant expression levels of both genes were detected in root tips and hypocotyls. The spatial distribution of GmODC and GmADC transcripts in primary and lateral roots and hypocotyls revealed that these genes are co-expressed in expanding cells of cortex parenchyma, expanding cells of central cylinder in main roots and in developing tissues and expanding cells of soybean hypocotyls. The data point out a correlation of the expression patterns of GmODC and GmADC gene to certain physiological roles such as organ development and cell expansion.

Induction and spatial organization of polyamine biosynthesis during nodule development in Lotus japonicus.

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an alpha-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

A Lotus japonicus beta-type carbonic anhydrase gene expression pattern suggests distinct physiological roles during nodule development.

A full-length cDNA clone, designated Ljca1, coding for a beta-type carbonic anhydrase (CA; EC: 4.2.1.1) was isolated from a Lotus japonicus nodule cDNA library. Semi-quantitative RT-PCR analysis revealed that Ljca1 codes for a nodule-specific CA, transcripts of which accumulate at maximum levels in young nodules at 14 days post-infection (d.p.i.). In situ hybridization and immunolocalization revealed that Ljca1 transcripts and LjCA1 polypeptides were present at high levels in all cell types of young nodules. In contrast, in mature nodules both transcripts and polypeptides were confined in a few cell layers of the nodules inner cortex. However, the central infected tissue of both young and mature nodules exhibited high CA activity, indicating the presence of additional CA isoforms of plant and/or microbial origin. This was supported by the finding that a putative Mesorhizobium loti CA gene was transiently expressed during nodule development. In addition, the temporal and spatial accumulation of phosphoenolpyruvate carboxylase (PEPC; EC: 4.1.1.31) was determined by semi-quantitative RT-PCR and immunolocalization. The results suggest that LjCA1 might fulfill different physiological needs during L. japonicus nodule development.

A sucrose transporter, LjSUT4, is up-regulated during Lotus japonicus nodule development.

LjSUT4, encoding a putative sucrose transporter, was identified in a Lotus japonicus nodule cDNA library. The deduced amino acid sequence showed a high degree of identity with sucrose transporters from other plants. Semi-quantitative RT-PCR analysis demonstrated that the L. japonicus SUT4 gene was expressed at high levels in both roots and nodules. In situ hybridization revealed that, in young nodules, SUT4 mRNA transcripts are present in vascular bundles, inner cortex and both infected and uninfected cells while, in mature nodules, accumulation of transcripts was restricted only in vascular bundles and the inner cortex. The results indicated that LjSUT4 codes for a putative sucrose transporter, and its expression pattern suggests a possible shift in the mechanism of sugar transport during nodule development. The role of this polypeptide in sucrose transport and metabolism is discussed.