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.

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.

Propagation of Casuarina equisetifolia through axillary buds of immature female inflorescences cultured in vitro.

The study of the actinorhizal symbiosis in Casuarina equisetifolia requires an homogenous plant material. Consequently, we devised a method of micropropagation based on the use of immature female inflorescences (IFI) as explants. IFI excised from an adult tree formed multiple buds after 4-week incubation on Murashige and Skoog medium with 0.05 µmol 1(-1) NAA and 11.1 µmol 1(-1) BAP. The axillary buds evolved into 5-6 cm long shoots 5 weeks after the transfer of IFI on a similar medium except for the addition of activated charcoal. Rooting of the shoots was obtained on a third medium, without BAP or charcoal, but with 1 µmol 1(-1) NAA. The plantlets were transferred into soil. Their growth was satisfactory and no plagiotropic tendency was observed.

[Organogenesis and Multiplication «in vitro¼ of Eucalyptus camaldulensis]. / Organogenèse et Multiplication «in vitro¼ chez l'Eucalyptus camaldulensis.

Organogenesis and shoot production have been achieved with Eucalyptus camaldulensis seedlings cultured on defined nutrient media supplemented with auxin (NAA) and cytokinin (BAP). Considerable shoot proliferation resulted from culturing isolating cotyledonary buds. Furthermore, callus generated buds without any prior transfer to alternate media. The technique described may be useful for improving and facilitating large scale cloning of selected plants.