Parmi les plantes superieures, l'association Rhizobium-legumineuses n'a pas l'exclusivite de la symbiose fixatrice d'azote. L'etude du modele Frankia-Casuarinacees, structurellement different du premier, mais aussi capable de fixer l'azote, pourrait apporter de nouvelles informations sur l'origine et l'evolution de ce type de symbioses.

In response to infection by Frankia, cells of Alnus glutinosa and Casuarina glauca nodules strongly express a subtilase-gene (ag12 and cg12 respectively). In order to gain a better understanding of the function of these genes in the symbiotic process, we have analysed the gene expression of ara12, an Arabidopsis thaliana homologue of cg12 and ag12. Transcription fusion of the promoter region of ara12 to the β-glucuronidase gene was introduced into A. thaliana. Para12-gus expression was found in young developing tissues. This result suggests that ara12 might be involved in proteins or polypeptides processing during A. thaliana development. We therefore speculate that cg12 and ag12, the actinorhizal homologues of ara12 might be involved in differentiation of Frankia infected cells in nodules.

A chimeric gene consisting of the β-glucuronidase (gusA) reporter gene under the control of the metallothionein-like promoter cgMT1 from the tropical tree Casuarina glauca was introduced into Nicotiana tabacum via Agrobacterium tumefaciens and into Oryza sativa by particle bombardment. The strongest histochemical staining for GUS activity was observed in the root system of the transgenic plants, and especially in lateral roots. In contrast, a relatively low level of reporter gene expression was seen in the aerial tissues and GUS staining was located mainly in the plant vascular system. The average ratio of GUS activity between root and leaf was found to be 13:1 in tobacco and 1.5:1 in rice. The pattern of cgMT1 promoter activity in floral organs was found to be different in tobacco and rice. High levels of gusA gene expression were detected in the ovules, pollen grains and tapetum, whereas in rice PcgMT1 directs expression to the vascular system of the floral organs. These results suggest that PcgMT1 is potentially useful in molecular breeding to express genes of interest whose products are preferentially needed in roots.

In response to infection by Frankia, cells of Alnus glutinosa and Casuarina glauca nodules strongly express a subtilase-gene (ag12 and cg12 respectively). In order to gain a better understanding of the function of these genes in the symbiotic process, we have analysed the gene expression of ara12, an Arabidopsis thaliana homologue of cg12 and ag12. Transcription fusion of the promoter region of ara12 to the β-glucuronidase gene was introduced into A. thaliana. Para12-gus expression was found in young developing tissues. This result suggests that ara12 might be involved in proteins or polypeptides processing during A. thaliana development. We therefore speculate that cg12 and ag12, the actinorhizal homologues of ara12 might be involved in differentiation of Frankia infected cells in nodules.

SummaryThe purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO3 and KNO3 was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO3 reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.

Transgenic Casuarinaceae and reporter genes provide valuable tools to study gene expression in transgenic actinorhizal nodules. In this paper, we discuss the use of ß-glucuronidase for the histochemical localization and quantification of gene expression in transgenic plants of Allocasuarina verticillata and Casuarina glauca nodulated by the actinomycete Frankia. We also report on the genetic transformation of A. verticillata by the Agrobacterium tumefaciens strain C58C1(pGV2260) containing the 35S-mgfp5-ER construct encoding a modified green fluorescent protein of Aequorea victoria in a binary vector. The evolution of the GFP fluorescence was monitored through all stages of the regeneration process. The data indicate that GFP is not toxic in Casuarinaceae and that this reporter gene can be used for visual screening of transformed calli and transgenic plants. The fluorescence pattern of gfp provides a new tool for monitoring in vivo transgene expression in actinorhizal plants.

A clone for a type 1 metallothionein (cgMT1) was isolated from a Casuarina glauca nodule cDNA library. The corresponding gene belongs to a small family and is highly expressed in roots and nitrogen-fixing nodules, whereas low expression was observed in aerial parts of the plant. The promoter region of cgMT1 was isolated and fused to the β-glucuronidase (gus) gene. Transgenic Casuarinaceae plants showed that the cgMT1 promoter was most active in roots and in the oldest region of the shoot. In situ hybridization indicated that in nodules cgMT1 transcript is present in mature Frankia-infected cells and in the pericycle. Possible roles for cgMT1 in symbiotic and non-symbiotic tissues are discussed.

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

•  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.

La source ultime de la diversite biologique reside dans la variabilite inscrite dans le patrimoine genetique des organismes. La diversite genetique naturelle, caracteristique de tout etre vivant, permet a une espece de s’adapter et de repondre a des changements de son environnement. Ainsi, l’adaptabilite de chaque individu determinee par son genotype est exprimee par une certaine plasticite phenotypique morpho-physiologique. Nous proposons d’envisager la diversite genetique au niveau d’un organe, la racine, au travers de quelques exemples pris chez les Angiospermes. Les exemples qui suivent permettent d’illuster comment une variation genetique au niveau moleculaire conduit aux modifications physiologiques de la racine lorsqu’elle est soumise aux contraintes biotiques et abiotiques de son milieu. Dans la legumineuse tropicale Sesbania rostrata, les ebauches racinaires de la tige, en position adventive, sont capables d’evoluer selon leur environnement en nodules fixateurs d’azote en presence de l’Azorhizobium specifique, en racine adventive typique ou bien en bourgeon adventif, lorsqu’un segment de tige est place en culture in vitro. Chez plusieurs especes de la famille des Casuarinacees, le systeme racinaire peut former des structures tres diverses selon leur environnement. Au contact de l’actinomycete specifique Frankia, des nodules fixateurs d’azote (actinorhizes) sont inities, en presence d’une carence en phosphore et en fer, des racines en touffes « cluster roots » se developpent et, enfin, en presence de champignons endo ou ectomycorhiziens, des structures nouvelles apparaissent qui sont les mycorhizes. Les resultats obtenus ces dernieres annees chez les legumineuses suggerent que certains genes communs de la plante hote seraient impliques a la fois dans l’interaction mycorhizienne et l’association plante-Rhizobium, suggerant par la le partage d’une voie de transduction commune dans les deux types d’interaction. Enfin, le regroupement des plantes pouvant developper des nodules fixateurs d’azote au sein d’un meme clade permet de penser que cet ensemble constitue une veritable ressource genetique au sens classique du terme, appliquee ici a la plasticite phenotypique d’un organe, la racine.