Katharina Pawlowski

Symbiotic Nitrogen Fixation.

that are expressed before the onset of nitrogen fixation and are involved in infection and nodule development. The products of the late nodulin genes are involved in the interaction with the endosymbiont and in the metabolic specialization of the nodule (Nap and Bisseling, 1990). In the first part of this review, we describe the early steps of the interaction between rhizobia and legumes that result in the formation of a nitrogen-fixing nodule. We focus on the role of specific lipooligosaccharides secreted by rhizobia in the induction of these early steps. In the second part, we describe nodule functioning and compare actinorhizal and legume nodules.

Rhizobial and Actinorhizal Symbioses: What Are the Shared Features?

In endophytic symbioses, microorganisms live intercellularly or intracellularly inside a plant, in a mutually beneficial rela? tionship. The host plant (the macrosymbiont) usually supplies the endophyte (the microsymbiont) with photosynthates, whereas the benefit of these associations for the plant is an improvement in access to important mineral nutrients whose supply would otherwise be growth limiting. With the exception of cyanobacterial symbioses, in most endophytic symbioses involving higher plants, microorganisms colonize the plant root (reviewed in Quispel, 1992). In mycorrhizal sym? bioses, the fungus improves the absorption of minerals, particularly phosphorus, by the roots (Read et al., 1992; Harrison and van Buuren, 1995; see also Gianinazzi-Pearson, 1996, in this issue). In root nodule symbioses involving eubacteria of the family Rhizobiaceae or actinomycetes of the genus Frankia, the microsymbiont supplies the plant host with am? monium, the product of biological nitrogen fixation. To provide an ecological niche for the endophyte, the mac? rosymbiont has to undergo symbiosis-specific differentiation. In nitrogen-fixing root nodule symbioses, the microsymbionts are hosted in special organs, the root nodules, which develop on roots upon infection by the microsymbiont (reviewed in Benson and Silvester, 1993; Mylona et al., 1995). In root sym? bioses with fungi, plant differentiation is less dramatic. In ectomycorrhizal symbioses, for example, the fungus somewhat alters root growth, triggering the formation of short thick infected roots (Martin and Hilbert, 1991), whereas in endomycorrhizal symbioses, the plant root structure is altered at the cellular level but not macroscopically (Koide, 1991; Gianinazzi-Pearson, 1996, in this issue). In this review, we discuss the recent progress in research on endophytic symbioses involving plant roots. Special attention is given to nitrogen-fixing symbiotic interactions, and we summarize our current knowledge of nodule formation and function. The last part of this review focuses on the develop? ment of model systems and the relationships between different endophytic symbioses involving plant roots.

Piriformospora indica affects plant growth by auxin production

Piriformospora indica has been shown to improve the growth of many plant species including Arabidopsis thaliana, but the mechanism by which this is achieved is still unclear. Arabidopsis root colonization by P. indica was examined in sterile culture on the medium of Murashige and Skoog. P. indica formed intracellular structures in Arabidopsis root epidermal cells and caused changes in root growth, leading to stunted and highly branched root systems. This effect was because of a diffusible factor and could be mimicked by IAA. In addition, P. indica was shown to produce IAA in liquid culture. We suggest that auxin production affecting root growth is responsible for, or at least contributes to, the beneficial effect of P. indica on its host plants.

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N2-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O2 flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N2-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.

A Nodule-Specific Dicarboxylate Transporter from Alder Is a Member of the Peptide Transporter Family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a Km of 70 μm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.

Genome Sequence of “Candidatus Frankia datiscae” Dg1, the Uncultured Microsymbiont from Nitrogen-Fixing Root Nodules of the Dicot Datisca glomerata

Members of the noncultured clade of Frankia enter into root nodule symbioses with actinorhizal species from the orders Cucurbitales and Rosales. We report the genome sequence of a member of this clade originally from Pakistan but obtained from root nodules of the American plant Datisca glomerata without isolation in culture.

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.

The diversity of actinorhizal symbiosis.

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.

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.

Asymmetric Responsiveness to Ethylene Mediates Cell Elongation in the Apical Hook of Peas

The apical hook of dark-grown dicotyledonous seedlings is a protective structure resulting from an inhibition of cell elongation on the inner portion of the hook. This differential growth response is mediated by ethylene. Expression of the gene encoding 1-aminocyclopropane-1-carboxylate oxidase (ACO), the terminal enzyme in ethylene biosynthesis, is induced by ethylene via a positive feedback loop. Therefore, the ACO transcript can serve as a molecular marker for both ethylene formation and ethylene responsiveness. We examined the distribution of ACO mRNA of pea, Ps-ACO1, and of ACO enzyme activity in the apical hook of etiolated pea seedlings. In situ hybridization showed that cells on the inner, concave side of pea hooks accumulated more Ps-ACO1 mRNA than did cells on the outer, convex side. The distribution of ACO enzyme activity followed the same pattern. A direct correlation was observed between the cellular distribution of Ps-ACO1 mRNA, ACO enzyme activity, and the inhibition of cell elongation. Pea seedlings treated with a saturating concentration of ethylene still accumulated higher levels of the Ps-ACO1 transcript on the inner side of the apical hook, demonstrating an increased responsiveness to ethylene in this tissue. These results indicate that an asymmetrically distributed component of the ethylene signal transduction pathway mediates hook formation. Based on existing genetic evidence, we propose that this component is downstream from the serine/threonine protein kinase CTR1.