Does ethylene mediate cluster root formation under iron deficiency?

Casuarina glauca develops proteoid (cluster) roots in response to Fe deficiency. This study set out to investigate the possible involvement of ethylene in the initiation and/or the morphogenesis of cluster roots (CR). For this purpose, the effect of Ag+ added as silver thiosulfate, an inhibitor of ethylene action has been studied in plants growing hydroponically. No CR formation was observed in these growth conditions. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine, 1- aminoisobutyric acid, aminoxyacetic acid or cobalt chloride also eliminated the positive effect of Fe deficiency on CR formation in C. glauca. CR were not formed in Fe- deficient roots in the presence of ethylene inhibitors, suggesting a role for ethylene in the morphological responses to Fe deficiency. Interestingly, treatment of Casuarina plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid stimulated significantly the formation of CR, even if plants are supplied with Fe. However, this stimulation did not reach the level of CR obtained in Fe-deficient plants. These results suggest that an ethylene-mediated signalling pathway is involved in CR formation process in C. glauca.

Angiosperm Gymnostoma trees produce root nodules colonized by arbuscular mycorrhizal fungi related to Glomus.

• Structure and fungal composition is presented here for 'mycorrhizal' nodules of two angiosperms of the genus Gymnostoma (Casuarinaceae), G. deplancheanum and G. nodiflorum. These species are endemic to New Caledonia, where they grow on ultramafic soils. The mycorrhizal nodules, which are modified lateral roots invaded by an arbuscular mycorrhizal fungus, occur in addition to N2 -fixing nodules. • Techniques included PCR amplification of extracted DNA, for species identification, and histological studies to compare the developmental pathway of Gymnostoma mycorrhizal nodules with that of actinorhizal nodules. • The fungal DNA suggested that the strain belongs to the genus Glomus (Glomales). The endophytic mycelium also contained typical Glomus arbuscules and hyphal coils. Structurally, Gymnostoma mycorrhizal nodules are similar to those described in some Coniferales and in Caesalpinioideae trees of French Guyana. • The mycorrhizal nodules of G. deplancheanum and G. nodiflorum contain a fungus belonging to the Glomales. The role of the nodules might be linked to the ecological situation of the host plants, which are pioneers in exposed and rocky habitats.

Inoculation of Acacia mangium with Alginate Beads Containing Selected Bradyrhizobium Strains under Field Conditions: Long-Term Effect on Plant Growth and Persistence of the Introduced Strains in Soil.

The growth response of Acacia mangium Willd. to inoculation with selected Bradyrhizobium strains was investigated in two field trials in the Ivory Coast (West Africa). In the first trial (Anguededou), four provenances (i.e., trees originating from seeds harvested in different geographical areas) of A. mangium were inoculated with four Bradyrhizobium strains from different origins. Six months after being transplanted in the field, the heights of all inoculated trees showed a statistically significant increase of 9 to 26% compared with those of uninoculated trees, with the most effective strain being Aust 13c. After 19 months, the positive effect of inoculation on tree growth was confirmed. The effect of A. mangium provenance on tree growth was also highly significant. Trees from the Oriomo provenance of Papua New Guinea had a mean height that was 25% greater than those of other provenances. Analysis of variance showed a highly significant effect of interaction between strain and host provenance factors. Thus, most effective strain x provenance combinations could be proposed. Immunological identification of strains clearly showed that 90 to 100% of nodules from trees inoculated with three of the four Bradyrhizobium strains or from uninoculated trees contained exclusively Aust 13c 23 months after tree transplantation. This predominance of Aust 13c in nodules was still observed 42 months after tree transplantation. The second experiment (Port-Bouët), performed with a different soil, confirmed the long-term positive effect of Aust 13c on plant growth, its high competitive ability against indigenous strains, and its persistence in soil. Strain Aust 13c should thus be of great interest for inoculating A. mangium under a wide range of field conditions.

Inoculant made of encapsulated Frankia: assessment of Frankia growth within alginate beads.

The growth of Frankia cells within alginate beads was inhibited when the amount encapsulated exceeded 0.5 to 2.5 µg protein/ml of beads. Frankia growth was observed not only in the beads incubated in nutrient media (with of without combined N), but also in those incubated in air provided they retained enough nutrients. The results allow some recommendations to be made for the preparation of Frankia inoculants.

Coordinate increase in activity of proteinase subunits of the 1300-kDa megaproteinase of Frankia strain BR: role of carbon source depletion and extracellular metabolites.

We have recently described the presence of a high molecular mass multicatalytic proteinase complex (megaproteinase; 28 S, 1300 kDa) in Frankia strain BR. The complex dissociates into 11 low molecular mass proteinase subunits (40-19 kDa) when subjected to sodium dodecyl sulfate - gelatin - polyacrylamide gel electrophoresis. We show here that the activity of these proteinase subunits strongly increased after cessation of growth in stirred BAP-PCM mineral medium. Subsequent addition of either BAP medium components or sodium propionate alone, as carbon source, to a Frankia culture at the end of the exponential growth phase was found to prolong growth for 1 additional day, and to delay the increase in activity of the proteinase subunits for 3 days after cessation of growth. Addition of ammonium chloride alone, as nitrogen source, had no effect. On the other hand, when Frankia cells in the late exponential phase (3 days) were resuspended in a culture filtrate recovered from a 5-day-old culture and supplemented with BAP-PCM medium components, the biomass yield decreased to about 50%. Also, the activity of the proteinase subunits increased as soon as growth ceased. The ability of this culture filtrate to inhibit growth and stimulate the activity of proteinase subunits was partially lost by heating or was largely removed by DEAE-cellulose treatment. Thus, our findings indicate an extracellular control of Frankia megaproteinase activity, suggesting that carbon source depletion and probably accumulation of heat-sensitive growth-inhibiting metabolites in the medium are determining factors.

High-molecular-mass multicatalytic proteinase complexes produced by the nitrogen-fixing actinomycete Frankia strain BR.

A major-high-molecular mass proteinase and seven latent minor proteinases were found in cell extracts and in concentrates of culture medium from Frankia sp. strain BR after nondenaturing electrophoresis in mixed gelatin-polyacrylamide gels. All of these complexes showed multicatalytic properties. Their molecular masses and their sedimentation coefficients varied from 1,300 kDa (28S) to 270 kDa (12S). The electroeluted 1,300-kDa proteinase complex dissociated into 11 low-molecular-mass proteinases (40 to 19 kDa) after sodium dodecyl sulfate activation at 30 degrees C and electrophoresis under denaturing conditions. All of these electroeluted proteinases hydrolyzed N-carbobenzoxy-Pro-Ala-Gly-Pro-4-methoxy-beta- naphthylamide, D-Val-Leu-Arg-4-methoxy-beta-naphthylamide, and Boc-Val-Pro-Arg-4-methyl-7-coumarylamide, whereas Suc-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide was cleaved only by the six lower-molecular-mass proteinases (27.5 to 19 kDa). Examination by electron microscopy of uranyl acetate-stained, electroeluted 1,300- and 650-kDa intracellular and extracellular proteinase complexes showed ring-shaped and cylindrical particles (10 to 11 nm in diameter, 15 to 16 nm long) similar to those of eukaryotic prosomes and proteasomes. Polyclonal antibodies raised against rat skeletal muscle proteasomes cross-reacted with all of the high-molecular-mass proteinase complexes and, after denaturation of the electroeluted 1,300-kDa band, with polypeptides of 35 to 38, 65, and 90 kDa. Electrophoresis of the activated cell extracts under denaturing conditions revealed 11 to 17 gelatinases from 40 to 19 kDa, including the 11 proteinases of the 1,300-kDa proteinase complex. The inhibition pattern of these proteinases is complex. Thiol-reactive compounds and 1-10-phenanthroline strongly inhibited all of the proteinases, but inhibitors against serine-type proteinases were also effective for most of them.

Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR.

To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.

Aerial Nodules in Casuarina cunninghamiana.

A complete survey of La Réunion Island showed that, in 40- to 50-year-old Casuarina cunninghamiana plantations located in the northeast at an altitude above 400 m, some trees bore aerial nodules as high as 6 to 7 m up the trunk. The nodules exhibited a significant specific acetylene reduction by the ARA method (0.77 mumol of C(2)H(4) per h/g [dry weight] of nodule) at the time of sampling (June 1990). Aerial nodules were also found on a Casuarina glauca trunk. Preliminary observations show that anatomically aerial and underground nodules do not differ significantly. In addition to host plant genetic determinants, aerial nodule formation is assumed to require sufficient rainfall, an abundance of Frankia spp. in the soil and air, and rhytidome on the tree trunk.

POlygalacturonic acid transeliminase production by Azospirillum species.

Polygalacturonic acid transeliminase (PATE) was produced by all of six Azospirillum strains studied. Characteristics were similar to those of PATE from other bacteria: activity was maximal at pH 8.0 and was stimulated by CaCl2. Polygalacturonic acid was used more readily than pectin as a substrate. Polygalacturonic acid in the medium stimulated PATE production by several but not all strains. In all cases some of the PATE produced in cultures remained bound to cell walls. In one strain, most remained cell wall bound. When nitrogen was supplied as amino acids rather than ammonium salts, the ratio of free to bound enzyme was increased. The strains studied varied considerably to response to nutrient amendments and in maximum PATE activity.

In vitro nitrogen fixation by two actinomycete strains isolated from casuarina nodules.

Acetylene reduction activity was demonstrated in pure cultures of two actinomycete strains isolated from nodules of Casuarina equisetifolia. This activity was comparable to that of free-living Rhizobium strains, but appeared to be less sensitive to pO(2) and more sensitive to the presence of combined nitrogen.

Polyacrylamide-entrapped Rhizobium as an inoculant for legumes.

Pot experiments showed that Rhizobium japonicum cells entrapped in a polyacrylamide gel could be used as an inoculant for soybeans and compared favorably to laboratory-made peat base inoculant containing the same bacterial strain.

Application of the fluorescent antibody technique to the study of an isolate of Beijerinckia in soil.

Fluorescent antibody was prepared against a temperate-soil isolate of Beijerinckia obtained from a rhizosphere of rice growing in Camargue (France). The antibody did not cross-react with any of 6 species of Azotobacter, 4 species of Beijerinckia, or 44 unidentified soil bacteria isolated from a spectrum of rhizospheres, but strongly stained the homologous Beijerinckia isolate. The isolate grew well in autoclave Camargue soil, but increased in numbers only slightly in nonsterile soil during 9 days. Preliminary examination of rice plants grown in the laboratory in soil from which the Beijerinckia was originally isolated did not show detectable Beijerinckia in the rhizosphere. The fluorescent antibody was sufficiently sensitive and specific to permit more extensive study of Beijerinckia in relation to nitrogen fixation in the rhizospher of rice.