C. Guan

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.

Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: expression of glutamine synthetase and acetylornithine transaminase.

Two nodule cDNA clones representing genes involved in Alnus glutinosa nitrogen metabolism were analysed. ag11 encoded glutamine synthetase (GS), the enzyme responsible for ammonium assimilation, while ag118 encoded acetylornithine transaminase (AOTA), an enzyme involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found at highest levels in root nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were found at high levels in nodules, confined to the infected cells, suggesting that in nodules of A. glutinosa, citrulline biosynthesis takes place mainly in the infected cells.

Distinct Patterns of Symbiosis-Related Gene Expression in Actinorhizal Nodules from Different Plant Families

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).

Gene expression in ineffective actinorhizal nodules of Alnus glutinosa.

Summary Several Frankia strains have been shown to induce ineffective, i.e. non-nitrogen fixing nodules, sometimes in a host-plant dependent manner. Previous studies have demonstrated that the resistance to nodulation of Alnus glutinosa by ineffective Frankia strains is genetically determined. In this study, ineffective nodules induced on susceptible Alnus glutinosa clones by soil suspensions from a local swamp were analysed cytologically. Comparisons with effective nodules showed that ineffective nodules contain higher amounts of polyphenols than effective nodules, indicating a plant defense reaction. Polyphenols were found even in the infected cortical cells. In situ hybridization with a Frankia antisense 16S rRNA probe showed that Frankia is degraded at an early stage of development of infected cells. The mRNAs of two plant genes, ag12ar\6 ag13, which had been found to be expressed in the infected cells of effective nodules, were localized in ineffective nodules. Their expression patterns seemed to be ...

Interaction between Frankia and actinorhizal plants.

A significant part of the nitrogen in living organisms is derived from atmospheric dinitrogen which gets incorporated into organic compounds by biological or chemical nitrogen fixation. Biological nitrogen fixation is an energy-consuming process performed by the enzyme nitrogenase, which is irreversibly denatured by oxygen. Nitrogenase is formed only by prokaryotes, which in some cases fix nitrogen in symbiosis with higher plants. Two groups of bacteria can enter symbioses with higher plants which lead to the formation of special organs, the root nodules, where the bacteria fix nitrogen while being hosted inside plant cells. The product of nitrogen fixation, ammonium, is exported to the plant, while the plant in turn provides its symbiont with energy sources. Azorhizobium, Bradyrhizobium, and Rhizobium enter symbioses with leguminous plants (with one exception, Parasponia from the Ulmaceae family; Trinick, 1979), and actinomycetes of the genus Frankia induce nodules on the roots of a diverse group of dicotyledonous plants, mostly woody shrubs, which are collectively referred to as actinorhizal plants (Benson and Silvester, 1993). While rhizobia are unicellular, Frankia usually grows in hyphal form, but can also form sporangia and, under certain conditions, nitrogen-fixing vesicles (Benson and Silvester, 1993). Vesicles are formed at the ends of hyphae or on short side branches either in the free-living state under nitrogen starvation and aerobic conditions, or in symbiosis. Within the vesicles, nitrogenase is protected from oxygen (Parsons et al., 1987) by the vesicle envelope, which consists of multiple layers of hopanoids, bacterial steroid lipids (Berry et al., 1993). In contrast to Frankia, rhizobia can use N2 as nitrogen source only in symbiosis (with one exception, Azorhizobium caulinodans ORS571; reviewed by de Bruijn, 1989).