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Microsymbionts are supplied with citrate in actinorhizal root nodules of Casuarina glauca and Datisca glomerata
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Two types of root nodule symbioses between nitrogen-fixing soil bacteria and higher plants are known, rhizobia/legume symbioses and the symbiosis between soil actinobacteria of the genus Frankia and more than 200 species from three different orders, collectively called actinorhizal plants. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen. Based on studies with bacterial mutants, it is known that rhizobia are supplied with dicarboxylates, specifically malate, but no legume malate exporters involved in the symbiosis have been identified thus far. For Frankia, a malate exporter from the NPF transporter family that locates to the perisymbiont membrane was identified in Alnus glutinosa (AgDCAT1; Jeong et al., 2004).   

In this study, the substrate specificity of two homologs of AgDCAT1 from nodules of two different actinorhizal hosts was analysed: CgDCAT1 from Casuarina glauca (Casuarinaceae, Fagales), a close relative of A. glutinosa, and DgDCAT1 from Datisca glomerata (Datiscaceae, Cucurbitales). A Multidrug And Toxic compound Extrusion (MATE) type protein from D. glomerata nodules (DgMATE1) was included in the analysis. The transcription of the corresponding genes of all three proteins was induced strongly in nodules compared to roots. Experiments using Xenopus laevis oocytes pre-loaded with 13C-labeled metabolites showed that all three proteins represent citrate exporters. Studies on the levels of citrate cycle intermediates showed that both citrate and malate were present at dramatically higher levels that 2‑oxoglutarate, succinate and fumate in roots and nodules of C. glauca as well as D. glomerata. The Casuarina-nodulating strain Frankia casuarinae CcI3 grew equally well on malate or citrate. In short, the results of this study led to the conclusion that intracellular Frankia bacteria are fed with citrate, not malate, in nodules of C. glauca and D. glomerata.

AgDCAT1 and CgDCAT1 represent orthologs, but DgDCAT1 maps to another subclade of the NPF family, showing that the NPF carboxylate exporters in actinorhizal nodules are polyphyletic. DgMATE1 represents a member of the detoxification (DTX) subgroup that seems to have been recruited for supplying Frankia with citrate. The data suggest that in different symbiotic lineages, carbon source exporters for the symbiosis were recruited from different transporter families independently.

National Category
Plant Biotechnology
Research subject
Plant Physiology
Identifiers
URN: urn:nbn:se:su:diva-172326OAI: oai:DiVA.org:su-172326DiVA, id: diva2:1346130
Available from: 2019-08-27 Created: 2019-08-27 Last updated: 2019-08-27Bibliographically approved
In thesis
1. The actinorhizal plant Datisca glomerata: interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms
Open this publication in new window or tab >>The actinorhizal plant Datisca glomerata: interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nitrogen is the element that most often limits plant growth and development. Common agricultural practices rely on the application of large quantities of industrially-produced nitrogen fertilizer, which poses a worldwide environmental threat. Sustainable agriculture encourages the use of biologically fixed nitrogen. However, access to this is still limited to a restricted group of dicotyledonous plants that share among them the ability to form a root nodule symbiosis. After an intricate molecular dialogue, these plants accommodate in the cells of a newly root-derived organ - the nodule - a class of bacteria that produce the nitrogenase enzyme by which they are able to reduce di-nitrogen from air to bioavailable ammonia. This mutualism allows the plant access to nitrogen in exchange for carbon. This thesis focuses particularly on the actinorhizal symbioses established between the North American plant Datisca glomerata (Datiscaceae, Cucurbitales) and Frankia actinobacteria from cluster II (Frankiaceae, Frankiales).

The main aim of this thesis was to improve our understanding about the genetic basis underlying the evolution of root nodule symbioses. Genome-wide comparative analysis indicated that the loss or fragmentation of genes coding for Nodule Inception (NIN) and/or Rhizobium-directed Polar Growth was a major event for the loss of nodulation in close relatives of plants that are able to form a root nodule symbiosis. To acquire more information about the requirements in plant adaptations to meet a symbiosis with Frankia cluster II strains, the nodule transcriptome of D. glomerata was compared with that of Ceanothus thyrsiflorus (Rhamnaceae, Rosales). This study suggested that cluster II Frankia strains use lipochitooligosaccharide Nod factors to signal to their host plants. In addition, it suggested that the nitrogen metabolism likely differs between these symbioses: while transcript profiles from nodules of D. glomerata supports pathways for arginine catabolism, which was previously suggested, those from nodules of C. thyrsiflorus support pathways for asparagine biosynthesis. Since nodules of both plants house Frankia strains from cluster II, the differences in nitrogen metabolism are most likely a feature of the host plant and not of the bacterial symbiont.

As part of an approach to establish D. glomerata as a model organism for actinorhizal Cucurbitales, the effects of phytohormones towards expression of genes putatively involved in signaling for nodule development were investigated. In D. glomerata, similarly to legume plants, the phytohormones cytokinin and auxin were proposed to play a central role in nodule development as they exert a positive effect on the expression of NIN as well as on that of genes whose promoters are presumably transactivated by NIN.

Furthermore, transporter proteins expressed in nodules of D. glomerata and of Casuarina glauca (Casuarinaceae, Fagales), which probably act in supplying C-metabolites to intracellular Frankia, were characterized for their substrate specificity. Results indicated that citrate, and not malate, might be the C-metabolite supplied to both Candidatus Frankia datiscae Dg1 and Frankia casuarinae CcI3 strains in symbiosis.

To explore the option of D. glomerata-mediated control towards its microsymbiont, a nodule-specific defensin-like peptide was characterized (DgDef1). Whereas DgDef1 acts as an antimicrobial peptide against Gram-negative strains in a range compatible with a role in symbiosis, no differentiation was shown in assays with the Gram-positive Streptomyces coelicolor. Nonetheless, DgDef1 induced changes in membrane integrity of the legume symbiont Sinorhizobium meliloti 1021 as well as in its transcription profile, e.g., on transcription of genes associated with dicarboxylate uptake. Thus, a role for DgDef1 in acting against ineffective microsymbionts is suggested. Phylogenetic analysis suggested that actinorhizal nodule-specific defensins and legume nodule-specific cysteine-rich peptides share a common origin, which in an evolutionary scenario of symbiont shift leads to the hypothesis that these peptides have been lost in most legumes lineages.

Collectively, the data presented in this thesis support the idea that root nodule symbioses share more mechanisms than previously assumed, e.g., in the defense against ineffective microsymbionts (“bacterial cheaters”), supporting the new paradigm that the common ancestor of legumes and actinorhizal plants had evolved a symbiosis that was later lost in most lineages.

Place, publisher, year, edition, pages
Stockholm University: Department of Ecology, Environment and Plant Sciences, Stockholm University, 2019. p. 62
Keywords
Root nodule symbiosis, nitrogen fixation, actinorhizal, Datisca glomer-ata, Frankia, nodule development, defensin, antimicrobial, carboxylate transporter, phylogenomics, transcriptomics
National Category
Other Biological Topics Botany
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-172315 (URN)978-91-7797-813-8 (ISBN)978-91-7797-814-5 (ISBN)
Public defence
2019-10-04, Vivi Täckholmssalen (Q-salen), Svante Arrhenius väg 20, Stockholm, 09:30 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2019-09-11 Created: 2019-08-27 Last updated: 2019-09-03Bibliographically approved

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