Casuarina glauca : a model tree for basic research in actinorhizal symbiosis

Casuarina glauca is a fast-growing multipurpose tree belonging to the Casuarinaceae family and native to Australia. It requires limited use of chemical fertilizers due to the symbiotic association with the nitrogen-fixing actinomycete Frankia and with mycorrhizal fungi, which help improve phosphorous and water uptake by the root system. C. glauca can grow in difficult sites, colonize eroded lands and improve their fertility, thereby enabling the subsequent growth of more demanding plant species. As a result, this tree is increasingly used for reforestation and reclamation of degraded lands in tropical and subtropical areas such as China and Egypt. Many tools have been developed in recent years to explore the molecular basis of the interaction between Frankia and C. glauca. These tools include in vitro culture of the host and genetic transformation with Agrobacterium, genome sequencing of Frankia and related studies, isolation of plant symbiotic genes combined with functional analyses (including knock-down expression based on RNA interference), and transcriptome analyses of roots inoculated with Frankia or Rhizophagus irregularis. These efforts have been fruitful since recent results established that many common molecular mechanisms regulate the nodulation process in actinorhizal plants and legumes, thus providing new insights into the evolution of nitrogen-fixing symbioses

Symbiotic signaling in actinorhizal symbioses

Actinorhizal symbioses are mutualistic associations between plants belonging to eight angiosperm families and soil bacteria of the genus Frankia. These interactions lead to the formation of new root organs, actinorhizal nodules, where the bacteria are hosted and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. It involves an elaborate signaling between both partners of the symbiosis. In recent years, our knowledge of this signaling pathway has increased tremendously thanks to a series of technical breakthroughs including the sequencing of three Frankia genomes [1] and the implementation of RNA silencing technology for two actinorhizal species. In this review, we describe all these recent advances, current researches on symbiotic signaling in actinorhizal symbioses and give some potential future research directions

Contribution of transgenic Casuarinaceae to our knowledge of the actinorhizal symbioses

The Casuarinaceae family is a group of 96 species of trees and shrubs that are tolerant to adverse soil and climatic conditions. In the field, Casuarinaceae bears nitrogen-fixing root nodules (so called actinorhizal nodules) resulting from infection by the soil actinomycete Frankia. The association between Casuarina and Frankia is of tremendous ecological importance in tropical and subtropical areas where these trees contribute to land stabilization and soil reclamation. During differentiation of the actinorhizal nodule, a set of genes called actinorhizal nodulins is activated in the developing nodule. Understanding the molecular basis of actinorhizal nodule ontogenesis requires molecular tools such as genomics together with gene transfer technologies for functional analysis of symbiotic genes. Using the biological vectors Agrobacterium rhizogenes and A. tumefaciens, gene transfer into the two species Allocasuarina verticillata and Casuarina glauca has been successful. Transgenic Casuarinaceae plants proved to be valuable tools for exploring the molecular mechanisms resulting from the infection process of actinorhizal plants by Frankia

The independent acquisition of plant root nitrogen-fixing symbiosis in Fabids recruited the same genetic pathway for nodule organogenesis

Only species belonging to the Fabid clade, limited to four classes and ten families of Angiosperms, are able to form nitrogen-fixing root nodule symbioses (RNS) with soil bacteria. This concerns plants of the legume family (Fabaceae) and Parasponia (Cannabaceae) associated with the Gram-negative proteobacteria collectively called rhizobia and actinorhizal plants associated with the Gram-positive actinomycetes of the genus Frankia. Calcium and calmodulin-dependent protein kinase (CCaMK) is a key component of the common signaling pathway leading to both rhizobial and arbuscular mycorrhizal symbioses (AM) and plays a central role in cross-signaling between root nodule organogenesis and infection processes. Here, we show that CCaMK is also needed for successful actinorhiza formation and interaction with AM fungi in the actinorhizal tree Casuarina glauca and is also able to restore both nodulation and AM symbioses in a Medicago truncatula ccamk mutant. Besides, we expressed auto-active CgCCaMK lacking the auto-inhibitory/CaM domain in two actinorhizal species: C. glauca (Casuarinaceae), which develops an intracellular infection pathway, and Discaria trinervis (Rhamnaceae) which is characterized by an ancestral intercellular infection mechanism. In both species, we found induction of nodulation independent of Frankia similar to response to the activation of CCaMK in the rhizobia-legume symbiosis and conclude that the regulation of actinorhiza organogenesis is conserved regardless of the infection mode. It has been suggested that rhizobial and actinorhizal symbioses originated from a common ancestor with several independent evolutionary origins. Our findings are consistent with the recruitment of a similar genetic pathway governing rhizobial and Frankia nodule organogenesis

Intraspecies variation in sodium partitioning, potassium and proline accumulation under salt stress in <em>Casuarina equisetifolia</em> Forst

International audienceCasuarina equisetifolia Forst., a member of the Casuarinaceae family, is widely planted in coastal areas due to its ability to function as potential barrier against wind and waves. Significant variation has been reported in the ability of C. equisetifolia to grow under salinity stress. In the present study, 82 clones of C. equisetifolia were assessed for their response to 50 mM incremental NaCl concentrations ranging from 50 mM to 550 mM in Hoagland's solution and clones with contrasted salt tolerance were identified. Several earlier reports attribute salt sensitivity in Casuarina species to the toxic effect of sodium. Intraclonal variation in the levels of sodium accumulation was therefore analysed. However, sodium content in the shoots and roots, showed little correlation (0.351 and -0.171) with salt tolerance in C. equisetifolia. Similarly, sodium to potassium ratio in the shoots and roots of NaCl treated and untreated clones also did not show correlation with mortality although certain tolerant clones exhibited selectivity of potassium over sodium under salt stress. Analysis of the shoot to root ratio of sodium however, showed better correlation (0.448) with salt tolerance, suggesting that restricted translocation of sodium to shoots and its relative retention in roots might play a crucial role in the salt tolerant clones of C. equisetifolia, and that shoot to root ratio of sodium could be a better parameter for salt tolerance in C. equisetifolia clones. The higher salt tolerance observed in certain clones despite higher sodium accumulation or shoot to root ratio of sodium suggests the presence of different multiple adaptive mechanisms that may be operating in different clones to help protect the cells from the toxic effects of sodium. The tolerant clone, TNIPT 4, which accumulated high concentrations of Na+, had low shoot to root ratio of Na+, and also a higher constitutive as well as NaCl induced accumulation of the compatible osmolyte, proline. The study thus emphasizes the need for characterising the genetic components involved in sodium transport, proline metabolism and other mechanisms contributing to salinity tolerance. The identified clones with contrasted stress tolerance mechanisms would thus be a valuable resource for transcriptomic, proteomic and metabolomic exploration in addition to their utility for field evaluation in flooded and coastal saline tracts