Alnus Glutinosa Nodules Research Articles

Draft genome sequences of two Micromonospora strains isolated from the root nodules of Alnus glutinosa.

In this paper, the draft genomes of Micromonospora RTGN7 and RTP1Z1, derived from Alnus glutinosa root nodules, are reported. The assembly of RTGN7 is 6.6 Mbp, composed of 59 contigs, with an N50 of 321,872. RTP1Z1's assembly is 6.3 Mbp, composed of 151 contigs, with an N50 of 76,442 bp.

The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe–Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa.

The genomes of two nitrogen-fixing Frankia strains, Ag45/Mut15 and AgPM24, isolated from root nodules of Alnus glutinosa are described as representatives of a novel candidate species. Phylogenomic and ANI analyses confirmed that both strains are related to cluster 1 frankiae, and that both strains belong to a novel species. At 6.4 - 6.7 Mb, their genomes were smaller than those of other cultivated Alnus-infective cluster 1 strains but larger than that of the non-cultivated Alnus-infective cluster 1 Sp+ strain AgTrS that was their closest neighbor as assessed by ANI. Comparative genomic analyses identified genes essential for nitrogen-fixation, gene composition as regards COGs, secondary metabolites clusters and transcriptional regulators typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. There were 459 genes present in other cultivated Alnus-infective strains lost in the two genomes, spread over the whole of the genome, which indicates genome erosion is taking place in these two strains.

Draft Genomes of Symbiotic Frankia Strains AgB32 and AgKG'84/4 from Root Nodules of Alnus Glutinosa growing under Contrasted Environmental Conditions.

The genomes of two nitrogen-fixing Frankia strains, AgB32 and AgKG'84/4, were isolated from spore-containing (spore+) and spore-free (spore-) root nodules of Alnus glutinosa, but they did not sporulate upon reinfection. The two strains are described as representatives of two novel candidate species. Phylogenomic and ANI analyses indicate that each strain represents a novel species within cluster 1, with genome sizes of 6.3 and 6.7 Mb smaller than or similar to those of other cultivated Alnus-infective cluster 1 strains. Genes essential for nitrogen-fixation, clusters of orthologous genes, secondary metabolite clusters and transcriptional regulators analyzed by comparative genomic analyses were typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. Compared to other cultivated Alnus-infective strains with large genomes, those of AgB32 and AgKG'84/4 had lost 380 or 409 genes, among which one hup cluster, one shc gene and the gvp cluster, which indicates genome erosion is taking place in these two strains.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

Isolation and characterization of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia

Actinobacterial isolates randomly obtained on nitrogen-free BAP medium from surface sterilized root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia sampled from fields were reported. They were assigned on the basis of partial 16S rRNA sequences to Micromonospora, Nocardia and Streptomyces genera. The isolates have been screened for hydrolytic activities, indole acetic acid (IAA) and siderophores production, phosphate solubilization and antagonistic activities. Results suggest putative traits as plant growth promoting bacteria proprieties of the isolates that occur in unique association in root nodules of the three analysed actinorhizal host species.

Ag13 is expressed in Alnus glutinosa nodules in infected cells during endosymbiont degradation and in the nodule pericycle*

ag13 is expressed in Alnus glutinosa nodules in infected cells during endosymbiont degradation and in the nodule pericycle*

Identification of agthi1, whose product is involved in biosynthesis of the thiamine precursor thiazole, in actinorhizal nodules of Alnus glutinosa.

A cDNA clone, pAgthi1, encoding a homologue of yeast Thi4, which is involved in thiazole biosynthesis, was isolated from a library made from poly(A) RNA from actinorhizal nodules of Alnus glutinosa by differential screening with nodule and root cDNA, respectively. The corresponding gene, agthi1, was shown to be expressed at high levels in nodules and shoot tips of A. glutinosa, while having low expression levels in roots, flowers, and developing fruits. The function of AgThi1 was demonstrated by yeast complementation studies, in which AgThi1 was able to rescue a yeast thi4 mutant when fused to the yeast Thi4 signal peptide. In A. glutinosa nodules, high levels of agthi1 mRNA were detected in the infected cortical cells and in the pericycle of the nodule vascular system. A homologue of this cDNA, ara6/tz, was identified in Arabidopsis thaliana. ara6 maps in a region of chromosome 5 of Arabidopsis containing the tz locus which is consistent with the fact that ara6 transcription is disturbed in two tz mutant lines. ara6/tz is expressed at high levels in chloroplast-containing parenchymatic cells of leaves, inflorescence shoots and flowers of Arabidopsis, and at lower levels in the vascular system.

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A nodule-specific gene encoding a subtilisin-like protease is expressed in early stages of actinorhizal nodule development.

To identify genes specifically expressed during early stages of actinorhizal nodule development, a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA, respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization, two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them, ag12 (A. glutinosa), was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganisms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi, and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals, subtilases are responsible for processing peptide hormones. A homolog of ag12, ara12, was identified in Arabidopsis; it was expressed in all organs, and its expression levels were highest during silique development. Hence, our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development.

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.

Occurrence of hemoglobin in the nitrogen‐fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia, the nitrogen‐fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O2 transport.

A Hypervariable 23S rRNA Region Provides a Discriminating Target for Specific Characterization of Uncultured and Cultured Frankia

Summary A highly variable part in domain III of the 23S rRNA specific for Gram-positive bacteria with a high DNA G+C content was evaluated as probe/target system for the specific detection of the actinomycete Frankia . Comparative sequence analysis of PCR amplified and cloned inserts from 35 Frankia strains confirmed the separation of these strains into host infection groups. The Casuarina and Elaeagnus groups were characterized by no or only small sequence variation. The Alnus group could be separated roughly into four subgroups, three containing typical nitrogen-fixing strains and a fourth one containing only non-nitrogen-fixing strains. To the latter group, two other non-infective and non-nitrogen-fixing strains, Cn3 and PtI4 isolated from Coriaria nepalensis and Purshia tridentata , respectively, could be aligned. The uncultured nodular endophyte of Coriaria nepalensis appeared well separated from all other sequences. The results of hybridization experiments with in vitro transcripts, PCR amplification products and oligonucleotides indicated a sufficient ratio of variability and stability within this 23S rRNA insertion to serve as target for subgroup-specific detection of Frankia within the Alnus group. Reliable discrimination with large probes like transcripts or PCR products, however, was only achieved between the non-nitrogen fixing Frankia strains and the nitrogen-fixing strains of the Alnus host infection group. The subgroups of the nitrogen-fixing strains were subsequently distinguished by oligonucleotide probes. Uncultured Frankia populations in root nodules of Alnus glutinosa could be characterized by these probes after PCR assisted sequence retrieval of the 23S rRNA insertion and cloning into E. coli .

Phylogenetic studies on uncultured Frankia populations in nodules of Datisca cannabina.

Part of the 16S rRNA gene was amplified directly from uncultured endophyte populations within the root nodules of Datisca cannabina and three strains isolated from nodules of Alnus glutinosa (AgKG'84/4), Coriaria nepalensis (Cn3), and D. cannabina (Dc2). Sequence comparison based on 930 nucleotides indicated that the endophyte of D. cannabina nodules belongs to the genus Frankia and is highly related to the endophyte of C. nepalensis root nodules. The relatedness of the endophytes of C. nepalensis and D. cannabina nodules was also reflected by closely related nifH sequences amplified from the nodules. 16S rRNA sequence analysis of the noninfective strains obtained from both D. cannabina (Dc2) and C. nepalensis (Cn3) nodules also revealed the close relationship of these strains to the genus Frankia.

Diversity of Frankia strains isolated from single nodules of Alnus glutinosa

Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.

Identification of Dipterocarpol as Isolation Factor for the Induction of Primary Isolation ofFrankiafrom Root Nodules ofAlnus glutinosa(L.) Gaertner

Identification of Dipterocarpol as Isolation Factor for the Induction of Primary Isolation of<i>Frankia</i>from Root Nodules of<i>Alnus glutinosa</i>(L.) Gaertner

The role of lipids in the growth of Frankia isolates

A comparison is made of the lipid requirements of some Frankia strains. Root-extract lipids from alder were essential for the isolation of Frankia strains from all nodules of Alnus glutinosa, Hippophaë rhamnoides, and in one case of Myrica gale and, at least in the case of A. glutinosa, they cannot be replaced by lecithin, Tween 80, or propionate. Many strains no longer need these lipids during subsequent cultivation. Strains from the spore-negative nodules of A. glutinosa need the lipids even after prolonged cultivation on most media, though they can adapt to media with propionate but without root-extract lipids after several transfers. These adapted strains must be distinguished from occasional revertants which, moreover, have a reduced infectivity. The results of silica-gel column and thin-layer chromatographic analysis combined with alkaline hydrolysis indicate that the active factors in the root-extract lipids are special fatty acids either in free or in bound form.

ABSCISIC ACID IN THE NODULATED ROOT SYSTEM OF ALNUS GLUTINOSA

SUMMARYEvidence for the presence of abscisic acid (ABA) in the perennial root nodules of Alnus glutinosa (L.) Gaertn. has been obtained by electron‐capture‐detection gas chromatography (GC–ECD), combined gas‐chromatography mass spectrometry (GC‐MS) and GC‐MS using single‐ion monitoring (GC‐SIM). The free‐ABA content of the nodules was shown by GC‐ECD analysis to fluctuate seasonally over the range 101 to 879 ng g−1 fresh weight. No obvious correlations were found between the amount of ABA detected and rates of nodule growth. However, ABA pools in dormant nodules were up to 25 times higher than in those from actively growing plants.

Glutamine synthetase in alder (Alnus glutinosa) root nodules. Purification, properties and cytoimmunochemical localization

Chromatographic, kinetic and regulatory properties of glutamine synthetase (GS) were investigated in root nodules of Alnus glutinosa L. (Gaertn) grown in a culture chamber. By DEAE Sephacel column chromatography and polyacrylamide gel electrophoresis, a major form of the enzyme was identified. Molecular weight was about 360 000 for the native protein and 43 000 for the subunits. Optimum pH was about 7.3 and Km for glutamate and ATP were 0.9 mM and 0.5 mM, respectively. By immunofluorescent techniques GS was localized in the inner cortical cells but not in vesicles of alder root nodules.

Carbon metabolism of Frankia spp. in root nodules of Alnus glutinosa and Hippophaë rhamnoides

The occurrence and localization of enzymes involved in glycolysis, tricarboxylic acid cycle and glyoxylate cycle in root nodules of Alnus glutinosa (L.) Vill. and Hippophaë rhamnoides L. ssp. rhamnoides were studied. The following enzymes, catalyzing reversible steps in the glycolysis, were found in both the endophyte Frankia spp. and the plant cytosol of Alnus nodules: fructose‐1,6‐diphosphate aldolase, glyceralde‐hyde‐3‐phosphate dehydrogenase, phosphoglycerate kinase and enolase. The enzymes catalyzing irreversible steps in glycolysis, viz. hexokinase and pyruvate kinase, were detectable only in the plant cytosol. Similar results were obtained with nodule homogenates of Hippophaë. This indicates the absence of a complete glycolysis in the endophyte. Vesicle clusters of the nodule endophyte of Alnus contained various dehydrogenases of the tricarboxylic acid cycle and showed activity of glutamate oxaloacetate transaminase. Respiration studies showed that vesicle clusters take up oxygen when supplied with NAD, glutamate and malate together. No oxygen uptake was found when any of these compounds was omitted. Vesicle clusters from both Alnus and Hippophaë nodules showed no detectable activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Since these enzymes are known to be present in Frankia Avcll, when grown in a medium with Tween 80 as carbon source, it is suggested that the glyoxylate cycle enzymes are repressed in the root‐nodule symbioses.