The role of Frankia inoculation in casuarina plantations in China.

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation.

Recent advances in actinorhizal symbiosis signaling.

Nitrogen and phosphorus availability are frequent limiting factors in plant growth and development. Certain bacteria and fungi form root endosymbiotic relationships with plants enabling them to exploit atmospheric nitrogen and soil phosphorus. The relationships between bacteria and plants include nitrogen-fixing Gram-negative proteobacteria called rhizobia that are able to interact with most leguminous plants (Fabaceae) but also with the non-legume Parasponia (Cannabaceae), and actinobacteria Frankia, which are able to interact with about 260 species collectively called actinorhizal plants. Fungi involved in the relationship with plants include Glomeromycota that form an arbuscular mycorrhizal (AM) association intracellularly within the roots of more than 80% of land plants. Increasing numbers of reports suggest that the rhizobial association with legumes has recycled part of the ancestral program used by most plants to interact with AM fungi. This review focuses on the most recent progress made in plant genetic control of root nodulation that occurs in non-legume actinorhizal plant species.

Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca(2+) spiking and NIN gene expression in the actinorhizal plant Casuarina glauca.

Although it is now well-established that decorated lipo-chitooligosaccharide Nod factors are the key rhizobial signals which initiate infection/nodulation in host legume species, the identity of the equivalent microbial signaling molecules in the Frankia/actinorhizal association remains elusive. With the objective of identifying Frankia symbiotic factors we present a novel approach based on both molecular and cellular pre-infection reporters expressed in the model actinorhizal species Casuarina glauca. By introducing the nuclear-localized cameleon Nup-YC2.1 into Casuarina glauca we show that cell-free culture supernatants of the compatible Frankia CcI3 strain are able to elicit sustained high frequency Ca(2+) spiking in host root hairs. Furthermore, an excellent correlation exists between the triggering of nuclear Ca(2+) spiking and the transcriptional activation of the ProCgNIN:GFP reporter as a function of the Frankia strain tested. These two pre-infection symbiotic responses have been used in combination to show that the signal molecules present in the Frankia CcI3 supernatant are hydrophilic, of low molecular weight and resistant to chitinase degradation. In conclusion, the biologically active symbiotic signals secreted by Frankia appear to be chemically distinct from the currently known chitin-based rhizobial/arbuscular mycorrhizal signaling molecules. Convenient bioassays in Casuarina glauca are now available for their full characterization.

The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals.

Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Biological nitrogen fixation in non-legume plants.

BACKGROUND: Nitrogen is an essential nutrient in plant growth. The ability of a plant to supply all or part of its requirements from biological nitrogen fixation (BNF) thanks to interactions with endosymbiotic, associative and endophytic symbionts, confers a great competitive advantage over non-nitrogen-fixing plants. SCOPE: Because BNF in legumes is well documented, this review focuses on BNF in non-legume plants. Despite the phylogenic and ecological diversity among diazotrophic bacteria and their hosts, tightly regulated communication is always necessary between the microorganisms and the host plant to achieve a successful interaction. Ongoing research efforts to improve knowledge of the molecular mechanisms underlying these original relationships and some common strategies leading to a successful relationship between the nitrogen-fixing microorganisms and their hosts are presented. CONCLUSIONS: Understanding the molecular mechanism of BNF outside the legume-rhizobium symbiosis could have important agronomic implications and enable the use of N-fertilizers to be reduced or even avoided. Indeed, in the short term, improved understanding could lead to more sustainable exploitation of the biodiversity of nitrogen-fixing organisms and, in the longer term, to the transfer of endosymbiotic nitrogen-fixation capacities to major non-legume crops.

Casuarina in Africa: distribution, role and importance of arbuscular mycorrhizal, ectomycorrhizal fungi and Frankia on plant development.

Exotic trees were introduced in Africa to rehabilitate degraded ecosystems. Introduced species included several Australian species belonging to the Casuarinaceae family. Casuarinas trees grow very fast and are resistant to drought and high salinity. They are particularly well adapted to poor and disturbed soils thanks to their capacity to establish symbiotic associations with mycorrhizal fungi -both arbuscular and ectomycorrhizal- and with the nitrogen-fixing bacteria Frankia. These trees are now widely distributed in more than 20 African countries. Casuarina are mainly used in forestation programs to rehabilitate degraded or polluted sites, to stabilise sand dunes and to provide fuelwood and charcoal and thus contribute considerably to improving livelihoods and local economies. In this paper, we describe the geographical distribution of Casuarina in Africa, their economic and ecological value and the role of the symbiotic interactions between Casuarina, mycorrhizal fungi and Frankia.

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3.

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.

Transcriptomics of actinorhizal symbioses reveals homologs of the whole common symbiotic signaling cascade.

Comparative transcriptomics of two actinorhizal symbiotic plants, Casuarina glauca and Alnus glutinosa, was used to gain insight into their symbiotic programs triggered following contact with the nitrogen-fixing actinobacterium Frankia. Approximately 14,000 unigenes were recovered in roots and 3-week-old nodules of each of the two species. A transcriptomic array was designed to monitor changes in expression levels between roots and nodules, enabling the identification of up- and down-regulated genes as well as root- and nodule-specific genes. The expression levels of several genes emblematic of symbiosis were confirmed by quantitative polymerase chain reaction. As expected, several genes related to carbon and nitrogen exchange, defense against pathogens, or stress resistance were strongly regulated. Furthermore, homolog genes of the common and nodule-specific signaling pathways known in legumes were identified in the two actinorhizal symbiotic plants. The conservation of the host plant signaling pathway is all the more surprising in light of the lack of canonical nod genes in the genomes of its bacterial symbiont, Frankia. The evolutionary pattern emerging from these studies reinforces the hypothesis of a common genetic ancestor of the Fabid (Eurosid I) nodulating clade with a genetic predisposition for nodulation.

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection.

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.

SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria.

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host-symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant-fungal and plant-bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.

Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots.

The study of arbuscular mycorrhiza often requires the staining of fungal structures using specific dyes. Fluorescent dyes such as acid fuchsin and wheat germ agglutinin conjugates give excellent results, but these compounds are either hazardous or very expensive. Here, we show that a safer and inexpensive dye, Uvitex2B, can be efficiently used to stain intraradical fungal structures formed by the arbuscular mycorrhizal fungus Glomus intraradices in three plant species: carrot, Casuarina equisetifolia, and Medicago truncatula. The intensity and stability of Uvitex2B allow the acquisition of high-quality images using not only confocal laser scanning microscopy but also epifluorescence microscopy coupled with image deconvolution. Furthermore, we demonstrate that Uvitex2B and ß-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.

Infection-specific activation of the Medicago truncatula Enod11 early nodulin gene promoter during actinorhizal root nodulation.

The MtEnod11 gene from Medicago truncatula is widely used as an early infection-related molecular marker for endosymbiotic associations involving both rhizobia and arbuscular mycorrhizal fungi. In this article, heterologous expression of the MtEnod11 promoter has been studied in two actinorhizal trees, Casuarina glauca and Allocasuarina verticillata. Transgenic C. glauca and A. verticillata expressing a ProMtEnod11::beta-glucuronidase (gus) fusion were generated and the activation of the transgene investigated in the context of the symbiotic associations with the N-fixing actinomycete Frankia and both endo- and ectomycorrhizal fungi (Glomus intraradices and Pisolithus albus, respectively). ProMtEnod11::gus expression was observed in root hairs, prenodules, and nodules and could be correlated with the infection of plant cells by Frankia spp. However, no activation of the gus reporter gene was detected prior to infection or in response to either rhizobial Nod factors or the wasp venom peptide MAS-7. Equally, ProMtEnod11::gus expression was not elicited during the symbiotic associations with either ecto- or endomycorrhizal fungi. These observations suggest that, although there is a conservation of gene regulatory pathways between legumes and actinorhizal plants in cells accommodating endosymbiotic N-fixing bacteria, the events preceding bacterial infection or related to mycorrhization appear to be less conserved.

Zinc Hyperaccumulation in Plants: A Review.

Zinc is an essential microelement involved in many aspects of plant growth and development. Abnormal zinc amounts, mostly due to human activities, can be toxic to flora, fauna, and humans. In plants, excess zinc causes morphological, biochemical, and physiological disorders. Some plants have the ability to resist and even accumulate zinc in their tissues. To date, 28 plant species have been described as zinc hyperaccumulators. These plants display several morphological, physiological, and biochemical adaptations resulting from the activation of molecular Zn hyperaccumulation mechanisms. These adaptations can be varied between species and within populations. In this review, we describe the physiological and biochemical as well as molecular mechanisms involved in zinc hyperaccumulation in plants.

Chitotetraose activates the fungal-dependent endosymbiotic signaling pathway in actinorhizal plant species.

Mutualistic plant-microbe associations are widespread in natural ecosystems and have made major contributions throughout the evolutionary history of terrestrial plants. Amongst the most remarkable of these are the so-called root endosymbioses, resulting from the intracellular colonization of host tissues by either arbuscular mycorrhizal (AM) fungi or nitrogen-fixing bacteria that both provide key nutrients to the host in exchange for energy-rich photosynthates. Actinorhizal host plants, members of the Eurosid 1 clade, are able to associate with both AM fungi and nitrogen-fixing actinomycetes known as Frankia. Currently, little is known about the molecular signaling that allows these plants to recognize their fungal and bacterial partners. In this article, we describe the use of an in vivo Ca2+ reporter to identify symbiotic signaling responses to AM fungi in roots of both Casuarina glauca and Discaria trinervis, actinorhizal species with contrasting modes of Frankia colonization. This approach has revealed that, for both actinorhizal hosts, the short-chain chitin oligomer chitotetraose is able to mimic AM fungal exudates in activating the conserved symbiosis signaling pathway (CSSP) in epidermal root cells targeted by AM fungi. These results mirror findings in other AM host plants including legumes and the monocot rice. In addition, we show that chitotetraose is a more efficient elicitor of CSSP activation compared to AM fungal lipo-chitooligosaccharides. These findings reinforce the likely role of short-chain chitin oligomers during the initial stages of the AM association, and are discussed in relation to both our current knowledge about molecular signaling during Frankia recognition as well as the different microsymbiont root colonization mechanisms employed by actinorhizal hosts.

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca.

cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a beta-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.

cg12 expression is specifically linked to infection of root hairs and cortical cells during Casuarina glauca and Allocasuarina verticillata actinorhizal nodule development.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Comparison of nodule induction in legume and actinorhizal symbioses: the induction of actinorhizal nodules does not involve ENOD40.

Two types of root nodule symbioses are known for higher plants, legume and actinorhizal symbioses. In legume symbioses, bacterial signal factors induce the expression of ENOD40 genes. We isolated an ENOD40 promoter from an actinorhizal plant, Casuarina glauca, and compared its expression pattern in a legume (Lotus japonicus) and an actinorhizal plant (Allocasuarina verticillata) with that of an ENOD40 promoter from the legume soybean (GmENOD40-2). In the actinorhizal Allocasuarina sp., CgENOD40-GUS and GmENOD40-2-GUS showed similar expression patterns in both vegetative and symbiotic development, and neither promoter was active during nodule induction. The nonsymbiotic expression pattern of CgENOD40-GUS in the legume genus Lotus resembled the nonsymbiotic expression patterns of legume ENOD40 genes; however, in contrast to GmENOD40-2-GUS, CgENOD40-GUS was not active during nodule induction. The fact that only legume, not actinorhizal, ENOD40 genes are induced during legume nodule induction can be linked to the phloem unloading mechanisms established in the zones of nodule induction in the roots of both types of host plants.

Effect of lead on root growth.

Lead (Pb) is one of the most widespread heavy metal contaminant in soils. It is highly toxic to living organisms. Pb has no biological function but can cause morphological, physiological, and biochemical dysfunctions in plants. Plants have developed a wide range of tolerance mechanisms that are activated in response to Pb exposure. Pb affects plants primarily through their root systems. Plant roots rapidly respond either (i) by the synthesis and deposition of callose, creating a barrier that stops Pb entering (ii) through the uptake of large amounts of Pb and its sequestration in the vacuole accompanied by changes in root growth and branching pattern or (iii) by its translocation to the aboveground parts of plant in the case of hyperaccumulators plants. Here we review the interactions of roots with the presence of Pb in the rhizosphere and the effect of Pb on the physiological and biochemical mechanisms of root development.