A.D.L. Akkermans

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.

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.

Ineffective Frankia strains in wet stands of Alnus glutinosa (L.) Gaertn. in the Netherlands.

Nodulation experiments using Alnus glutinosa L. Gaertn. (black alder) seedlings as hosts and soil suspensions as inoculants were made to determine the presence and relative amounts of ineffective (non-nitrogen fixing but infectious) Frankia strains in 10 waterlogged soils of natural alder stands in the Netherlands. Ineffective nodules were formed with soil from six locations, while effective nodules were found at all sites. From three of these locations the majority of the nodules formed were of the ineffective type. These results suggested that ineffective strains form an important fraction of the Frankia population of wet soils under black alder vegetation. No correlation (r(2), P <0.05) was found between the distribution of these Frankia strains and single soil chemical components. [KEYWORDS: Root; compatibility; endophyte; nodules; host; soil]

Expression of Frankia nif genes in nodules of Alnus glutinosa

Expression of Frankia genes involved in nitrogen fixation was studied in Alnus glutinosa nodules using the in situ hybridization technique. The results show that high level expression of nif genes does not occur immediately upon infection of cortical cells by Frankia. Also, only in the infected cells near the tips of the nodule lobes, nif genes are expressed at high levels. In the majority of infected cells, nif gene expression is rather low.

Ineffective Frankia and host resistance in natural populations of Alnus glutinosa (L.) Gaertn

Alnus glutinosa (black alder) populations are known to exhibit a variable degree of incompatibility to root nodule formation by ineffective Frankia. The relationship between the occurrence of ineffective Frankia in wet stands of black alder and the degree of resistance to nodulation by ineffective Frankia of seed-lots and clones of alder trees from these particular locations was studied through soil inoculation experiments. The average percentage of resistant plants (R-frequency) among the seed-lots from locations with an ineffective Frankia soil population was equal to, or higher than, the R-frequencies of locations without ineffective Frankia. The mean R-frequency was highest for the seed-lots from the location from which the soil inoculant was taken. These results strongly suggest that ineffective Frankia are not strictly dependent on susceptible A. glutinosa for the maintenance of their population size. The fungus Penicillium nodositatum also nodulated A. glutinosa seedlings. Whereas a negative interaction with the ineffective Frankia nodulation was found, this did not have a significant effect on the R-frequencies of the seed-lots that were tested, suggesting that the ineffective Frankia nodulation adversely affected the myco-nodulation, and not vice versa.

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 ...