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.

Casuarina glauca prenodule cells display the same differentiation as the corresponding nodule cells.

Recent phylogenetic studies have implied that all plants able to enter root nodule symbioses with nitrogen-fixing bacteria go back to a common ancestor (D.E. Soltis, P.S. Soltis, D.R. Morgan, S.M. Swensen, B.C. Mullin, J.M. Dowd, and P.G. Martin, Proc. Natl. Acad. Sci. USA, 92:2647-2651, 1995). However, nodules formed by plants from different groups are distinct in nodule organogenesis and structure. In most groups, nodule organogenesis involves the induction of cortical cell divisions. In legumes these divisions lead to the formation of a nodule primordium, while in non-legumes they lead to the formation of a so-called prenodule consisting of infected and uninfected cells. Nodule primordium formation does not involve prenodule cells, and the function of prenodules is not known. Here, we examine the differentiation of actinorhizal prenodule cells in comparison to nodule cells with regard to both symbionts. Our findings indicate that prenodules represent primitive symbiotic organs whose cell types display the same characteristics as their nodule counterparts. The results are discussed in the context of the evolution of root nodule symbioses.

Characterization of a Casuarina glauca nodule-specific subtilisin-like protease gene, a homolog of Alnus glutinosa ag12.

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.

Flavan-containing cells delimit Frankia-infected compartments in Casuarina glauca nodules.

We investigated the involvement of polyphenols in the Casuarina glauca-Frankia symbiosis. Histological analysis revealed a cell-specific accumulation of phenolics in C. glauca nodule lobes, creating a compartmentation in the cortex. Histochemical and biochemical analyses indicated that these phenolic compounds belong to the flavan class of flavonoids. We show that the same compounds were synthesized in nodules and uninfected roots. However, the amount of each flavan was dramatically increased in nodules compared with uninfected roots. The use of in situ hybridization established that chalcone synthase transcripts accumulate in flavan-containing cells at the apex of the nodule lobe. Our findings are discussed in view of the possible role of flavans in plant-microbe interactions.

Hairy root nodulation of Casuarina glauca: a system for the study of symbiotic gene expression in an actinorhizal tree.

The purpose of this study was to establish a fast system for producing transgenic actinorhizal root nodules of Casuarina glauca. Agrobacterium rhizogenes strain A4RS carrying the p35S-gusA-int gene construct was used to induce hairy roots on hypocotyls of 3-week-old C. glauca seedlings. Three weeks after wounding, the original root system was excised, and composite plants consisting of transgenic roots on untransformed shoots were transferred to test tubes to be inoculated with Frankia. The actinorhizal nodules formed on transformed roots had the nitrogenase activity and morphology of untransformed nodules. beta-Glucuronidase (GUS) activity was examined in transgenic roots and nodules by fluorometric and histochemical assays. The results indicate that transgenic nodules generated with this root transformation system could facilitate the molecular study of symbiotic nitrogen fixation in actinorhizal trees.

Transient gene expression in cassava using high-velocity microprojectiles.

The bacterial gene encoding beta-glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter from Arabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3' ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.

Characterization of several tropical strains of Anabaena and Nostoc: morphological and physiological properties, and plasmid content.

Thirty Anabaena and Nostoc strains isolated from West African soils were characterized for their morphological and physiological properties, and surveyed for plasmids. Most strains were found to harbour one to five plasmids whose molecular masses ranged from 1.5 to 120-150 X 10(6) daltons. No relationship between these plasmids and phenotypic properties such as carbon source utilization, antibiotic resistance or cyanophage and toxin production could be established.

Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense.

Homology was detected between the structural genes for the nitrogenase complex of K. pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains. Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A. brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K. pneumoniae nif probe. Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K. pneumoniae nif genes was found to be approximately 5 kb. Proteins encoded by the AbRI fragment were examined after infection of E. coli minicells.

Physiological properties and plasmid content of several strains of Azospirillum brasilense and A. lipoferum.

Four strains of Azospirillum brasilense, including strain 7000 (ATCC 29145) and five strains of A. lipoferum, including strain Br17 (ATCC 29709) were examined for the presence of plasmids. All the strains were found to harbour 1 to 5 plasmids whose molecular weight ranged from 3.5 to over 300 Md. No obvious relationship between the plasmids and phenotypic properties was established as yet, in particular N2 fixation, substrate utilization, drug resistance and lysogenic state. Six out of the 9 strains were lysogenic and phage production was inducible by mitomycin C. An icosahedric phage was purified from strain 7000.