Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.     

Genotypic variation of the response to cadmium toxicity in Pisum sativum L

This work evaluates the (cor-)relations between selected biochemical responses to toxic Cd and the degree of Cd sensitivity in a set of pea genotypes. Ten genotypes were analysed that differ in their growth response to Cd when expressed as root or shoot tolerance indices (TIs). Concentrations of non-protein thiols (NPTs) and malondialdehyde (MDA), activity of chitinase, peroxidase (POX), and catalase significantly increased in all pea genotypes treated with Cd. Cd-sensitivity of genotypes was correlated with relative increases in MDA concentration as well as activities of chitinase and POX, suggesting similar Cd stress effects. Activities of ascorbate peroxidase (APX) decreased, but concentrations of glutathione (GSH) increased in the less Cd-sensitive genotypes. Differences in root and leaf contents of Cd revealed no correlation with TI, metabolic parameters, and enzyme activities in Cd-treated plants, respectively, except that shoot Cd concentration positively correlated with shoot chitinase activity. Toxic Cd levels inhibited uptake of nutrient elements such as P, K, S, Ca, Zn, Mn, and B by plants in an organ- and genotype-specific manner. Cd-sensitivity was significantly correlated with decreased root Zn concentrations. The results show both similarities, as well as distinct features, in Cd toxicity expression in genotypes of one species, suggesting that independent and multi-factorial reactions modulate Cd sensitivity on the low-tolerance level of plants. The study illustrates the biochemical basis of earlier detected genotypic variation in Cd response.     

Evidence for horizontal transfer of 1-aminocyclopropane-1-carboxylate deaminase genes

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.     

Selective, high-resolution fluorescence imaging of mitochondrial Ca2+ concentration.

We have developed a digital image processing technique based on highpass filtering of microfluorimetric images for selective transmission of fine image details corresponding to mitochondria. This technique enabled the detection of the mitochondrial calcium signals with high selectivity, simultaneously with the cytosolic calcium signal. The validity of this technique was supported in primary cultures of rat brain capillary endothelial cells loaded with X-rhod-1 by the results that (i) inhibition of the mitochondrial Ca2+ uptake by discharging the mitochondrial membrane potential selectively abolished the transient of the highpass filtered signal evoked by ATP, and (ii) CGP-37157, a selective blocker of the mitochondrial Na+/Ca2+ exchanger, increased the peak amplitude of highpass filtered (mitochondrial) Ca2+ transients and caused a sustained plateau. The highpass filtering technique enabled the analysis of the mitochondrial Ca2+ transients in high temporal resolution. We found a uniform and monophasic rise of [Ca2+] in the mitochondrial population of the cell, following the cytosolic [Ca2+] with a delay at onset and peak. The introduced highpass filtering technique is a powerful tool in the high spatial and temporal resolution analysis of mitochondrial calcium transients, and it could be especially important in specimens where genetically targeted probes fail.     

NaCl-preferring NZB/B1NJ mice and NaCl-avoiding CBA/J mice have similar amiloride inhibition of chorda tympani responses to NaCl

Integrated chorda tympani nerve responses to NaCl were studied in two mouse strains, an NaCl-preferring NZB/B1NJ and an NaCl-avoiding CBA/J. The NaCl responses of both strains had similar magnitude and were suppressed by amiloride to a similar extent. This suggests that peripheral gustatory responsiveness to NaCl is not the only mechanism underlying mouse strain variation in NaCl acceptance.      

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

INDUCTION OF ERYTHROID MATURATION BY DIMETHYL SULFOXIDE IN FRIEND LEUKEMIC CELLS

Enhancement of the erythroid maturation in Friend virus-induced leukemic cells has been examined in vitro by the treatment with dimethyl sulfoxide (DMSO). Although the cell growth was inhibited in the medium containing 2% DMSO, many cells remained viable for a week. By the 3rd day of the culture, the cells treated with DMSO became more strongly agglutinated by phytohemagglutinin than the cells incubated without DMSO. Mouse erythrocyte membrane-specific antigens were also detectable at the 4th day. At the 8th day of the culture hemoglobin synthesis was apparently demonstrated in the cells treated with DMSO, which could not be seen in the untreated cells. Maturation or differentiation along the erythroid pathway in Friend leukemic cells by DMSO is discussed on these markers.     

Evidence for Horizontal Transfer of 1-Aminocyclopropane-1-Carboxylate Deaminase Genes

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.     

Rhizobacteria that produce auxins and contain 1‐amino‐cyclopropane‐1‐carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well‐watered and water‐limited potato (Solanum tuberosum)

Plant‐growth‐promoting rhizobacteria (PGPR) utilise amino acids exuded from plant root systems, but hitherto there have been no direct measurements of rhizosphere concentrations of the amino acid 1‐amino‐cyclopropane‐1‐carboxylic acid (ACC) following inoculation with PGPR containing the enzyme ACC deaminase. When introduced to the rhizosphere of two potato (Solanum tuberosum) cultivars (cv. Swift and cv. Nevsky), various ACC deaminase containing rhizobacteria (Achromobacter xylosoxidans Cm4, Pseudomonas oryzihabitans Ep4 and Variovorax paradoxus 5C‐2) not only decreased rhizosphere ACC concentrations but also decreased concentrations of several proteinogenic amino acids (glutamic acid, histidine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine). These effects were not always correlated with the ability of the bacteria to metabolise these compounds in vitro, suggesting bacterial mediation of root amino acid exudation. All rhizobacteria showed similar root colonisation following inoculation of sand cultures, thus species differences in amino acid utilisation profiles apparently did not confer any selective advantage in the potato rhizosphere. Rhizobacterial inoculation increased root biomass (by up to 50%) and tuber yield (by up to 40%) in pot trials, and tuber yield (by up to 27%) in field experiments, especially when plants were grown under water‐limited conditions. Nevertheless, inoculated and control plants showed similar leaf water relations, indicating that alternative mechanisms (regulation of phytohormone balance) were responsible for growth promotion. Rhizobacteria generally increased tuber number more than individual tuber weight, suggesting that accelerated vegetative development was responsible for increased yield.