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A bioassay to analyze the expression of orthologues of legume genes involved in nodulation of the actinorhizal plant Datisca glomerata: auxins and cytokinin induce NIN expression
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Two types of nitrogen-fixing root nodules are known, legume nodules and actinorhizal nodules. During the induction of nitrogen-fixing root nodules, phytohormones are involved in regulating gene expression to shape essential processes. One example is the expression of the early nodulin NODULE INCEPTION (NIN), which in the model legume Lotus japonicus is induced by cytokinin. In order to develop a bioassay for a quick and superficial analysis how phytohormones affect the expression of nodulation-related genes in different root nodule-forming plants, an axenic liquid culture system was established. The bioassay was used to examine responses in roots of the actinorhizal plant Datisca glomerata to phytohormones using quantitative RT-PCR. The synthetic cytokinin 6-Benzylaminopurine (BAP), the natural auxin phenyl acetic acid (PAA), and the synthetic auxin 1-Naphthaleneacetic Acid (NAA) were used. Marker genes for auxins and cytokinin responses were identified. PAA induced the expression of DgSAUR1, whereas NAA induced DgGH3.1.

Induction of Lotus japonicus NIN was analysed for proof of concept, and the bioassay showed induction by cytokinin and auxin (NAA). D. glomerata NIN1 was induced by PAA, NAA, and more weakly by BAP. The gene encoding a transcription factor that activates NIN expression, CYCLOPS, was induced by PAA and NAA. To see whether induction of NIN1 expression led to the activation of target genes of NIN, orthologs of the transcription factors NF-YA1, NSP1 and NSP2, and of ERN1 were identified in D. glomerata and the transcription of the corresponding genes was analysed for regulation by BAP, PAA or NAA. Induction by PAA was found for all genes examined. Additionally, BAP and PAA were used individually or in combination with antagonists of ethylene or gibberellin biosynthesis, respectively. Both antagonists abolished the induction of NIN by BAP or PAA; furthermore, no induction of NIN was found when BAP and PAA were applied together.

Altogether, these results show that the bioassay system can be used to identify target genes of NIN in legumes and actinorhizal plants for further analysis of the role of phytohormones in nodule induction.

National Category
Plant Biotechnology
Research subject
Plant Physiology
Identifiers
URN: urn:nbn:se:su:diva-172324OAI: oai:DiVA.org:su-172324DiVA, id: diva2:1346127
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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