Nitrogen-fixing root nodule symbioses

 

Nitrogen is the element that most often limits plant growth. Only prokaryotes that can form the nitrogenase enzyme complex can reduce air dinitrogen to ammonia and thereby bring it from the atmosphere into the biosphere. Plants that can enter a root nodule symbiosis with nitrogen-fixing soil bacteria are independent of soil nitrogen, i.e. fertilizer. In root nodules, the microsymbionts are hosted within plant cells, fix dinitrogen and deliver the products of nitrogen fixation to the host plant, while the host plant supplies them with carbon sources. Only plants belonging to a particular group, the so-called Fabid clade (but not all of them), are able to establish a root nodule symbiosis. Two groups of microsymbionts are involved in these symbioses – rhizobia with legumes and with Parasponia sp. from the Cannabaceae family, and Frankia strains with actinorhizal plants. Actinorhizal plants - mostly trees or woody shrubs - belong to eight different families from three different orders (Fagales, Rosales and Cucurbitales; look here for details).

 

Evolution of root nodule symbioses

The question arises which common feature of these plants represents the precondition that allowed the evolution of a root nodule symbiosis. Therefore, we compare root nodule symbioses from different branches of the Fabid clade in order to identify the common vs. family-specific features. The wide phylogenetic range of actinorhizal plant means that there is significant diversity among symbioses involving host plants from different families (see here). We are working on different plant species:

 

Legumes:

Medicago truncatula - model legume, indeterminate nodules, intracellular infection by Sinorhizobium meliloti

Lotus japonicus - model legume, determinate nodules, intracellular infection by Mesorhizobium loti

 

Actinorhizal plants:

Casuarina glauca (swamp oak) - Australian tree, intracellular infection by Frankia cluster-1c

Alnus glutinosa (black alder) - Northern hemisphere tree, intracellular infection by Frankia cluster-1a

Coriaria spp. - shrubs, distributed in Mediterranean, Northern India/Pakistan/Nepal, China, Japan, Taiwan, Philippines, Papua New Guinea, New Zealand, South America, pathway of infection by Frankia cluster-2 under examination

Datisca glomerata (Durango root) - Northwest American suffruticose plant, pathway of infection by Frankia cluster-2 under examination

Ceanothus thyrsiflorus – Northwest American shrub, intercellular infection by Frankia cluster-2

 

On the microbsymbiont side, in contrast with rhizobia, Frankia strains are monophyletic. Phylogenetically, there are four Frankia clusters, three of which representing facultative symbionts while cluster IV contains only non-symbiotic strains. Genome sizes differ strongly between strains from different clusters (cluster I genomes are ca. 7.5 MB in size except for the subgroup that nodulates Casuarinaceae which with 5.3 – 5.7 MB shows stronger genome reduction, cluster II genomes (5.3-5.6 MB) also show strong genome reduction, while clusters III (9-10 MB) and IV (ca. 10 MB) have the largest genome). Host range is correlated with phylogeny – e.g., members of cluster I nodulate actinorhizal Fagales (with the exception of Gymnostoma sp. (Casuarinaceae) and Myrica sp. (Myricaceae), members of cluster II nodule actinorhizal Cucurbitales and from the Rosales the actinorhizal Rosaceae and Ceanothus sp. (Rhamnaceae). Members of cluster III nodulate actinorhizal members of two Rosales families (Elaeagnaceae and Rhamnaceae with the exception of Ceanothus sp.) and the outlier genera of the actinorhizal Fagales, Gymnostoma sp. and Myrica sp. However, as a rule a member of a particular cluster cannot nodulate all host plants of said cluster.

            Phylogenetically, cluster II is basal to all other Frankia clusters (see here). So far, only one member of this cluster could be cultured, and this one, in contrast with all other known Frankia strains, is alkaliphile (see here). We are particularly interested in the evolution of cluster II and its host plants.

 

Projects (people from SU underlined):

• Comparison of Frankia cluster II genomes from different areas (Fede Berckx, Hsiao-Han Lin, Cyndi Mae Bandong, Jessica Simbahan, Luis Gabriel Wall, Than Van Nguyen, Daniel Wibberg, Jörn Kalinowski)
• Control of the microsymbiont by the plant in nodules of Datisca glomerata: characterization of nodule-specific cysteine-rich peptides (Irina Demina, Marco Salgado)
• What is the carbon source provided by Datisca glomerata to its microsymbiont? (Marco Salgado, Irina Demina, Fede Berckx, Nadia Binte Obaid, Sabine Zimmermann)
• Mechanism of stable internal accommodation of Frankia in actinorhizal nodules of Casuarina glauca and Datisca glomerata (Behnoosh Rashidi, Kirill Demchenko)
• Conservation of transcription factors involved in nodule development: activity of nodule-specific promoters in heterologous systems (Behnoosh Rashidi, Sara Mehrabi, Kirill Demchenko, Patricia Santos, Beth Mullin)
• Differentiation of infected cells in Datisca glomerata: transcriptome analysis using laser capture microdissection (Nadia Binte Obaid, Irina Demina, Marco Salgado, Nicole Gaude, Franziska Krajinski, Max Griesmann, Christoph Ziegenhain, Wolfgang Enard)
• Comparison of nodule development and differentiation in two host plants of cluster-2 Frankia, Datisca glomerata and Ceanothus thyrsiflorus (Thanh Van Nguyen, Marco Salgado, Fede Berckx, Rolf Hilker, Daniel Wibberg, Jörn Kalinowski, Kai Battenberg, Alison Berry)
• Metabolomics of actinorhizal nodules (Tomas Persson, Thanh Van Nguyen, Fede Berckx, Stefano Papazian, Benedicte Albrectsen, Alison Berry, Nicole Alloisio, Philippe Normand)
• Auxin and Datisca glomerata nodules (Irina Demina, Pooja Jha Maity, Anurupa Nagchowdhury, Marco Salgado, Ulrike Mathesius)

 

Group members (current):

• Nadia Binte Obaid (PhD student)
• Ciara Morrison (MSc student)
• Matilda Sandin (MSc student)

 

Former group members:

• Anna Maria Zdyb (PhD) (now: Institute of Genetics, Technical University Dresden, Zellescher Weg 20b, 01062 Dresden, Germany)
• Małgorzata Płaszczyca (PhD) (now: Rybnik, Poland)
• Patricia Santos (PostDoc) (now: Department of Biochemistry and Molecular Biology, University of Nevada, Reno 89557, NV, USA)
• Behnoosh Rashidi (PhD) (now: Copenhagen, Denmark)
• Sara Mehrabi (MSc) (now: Department of Ecology and Genetics, Evolutionary Biology, Norbyvägen 18D, 752 36 Uppsala, Sweden)
• Tomas Persson (PhD) (now: Department for Mathematics and Science Education, Stockholm University)
• Stefano Papazian (MSc) (now: Exposomics, SciLifeLab, Stockholm, Sweden)
• Irina V. Demina (PhD) (now: patent lawyer at BRANN AB, Uppsala, Sweden)
• Anurupa Nagchowdhury (MSc) (now: Karolinska Institutet, Stockholm, Sweden)
• Pooja Jha Maity (PostDoc) (Department of Botany, Ramjas College, University of Delhi, Delhi 110 007, India)
• Xiaoyun Gong (MSc) (now: Genetics, LMU Munich, Biocenter, Großhaderner Str. 4, 82152 Martinsried, Germany)
• Thanh Van Nguyen (PhD) (Novogene Europe, Uppsala, Sweden)
• Marco Guedes Salgado (PhD) (now: Institute of Biotechnology HiLIFE, Helsinki University, Viikinkaari 5d, 00790 Helsinki, Finland)
• Nadia Binte Obaid (MSc) (now: PhD student in the same group)
• Emilia Regazzoni (MSc) (now: Research Engineer at SciLifeLab, Solnavägen 9, Biomedicum, 17165 Solna, Sweden)
• András Patyi (MSc) (now: Department of Crop Sciences, FIBL, Ackerstrasse 113, CH-5070 Frick, Switzerland
• Fede Berckx (PhD) (now: Department of Crop Production Ecology, SLU Uppsala, Ekologicentrum, Ulls väg 16, 756 51 Uppsala, Sweden)

 

Funding (currently):

VR

 

External collaborations:

• Paul Dahlin (Agroscope, Research Division, Plant Protection, Phytopathology and Zoology in Fruit and Vegetable
  Production, 8820 Wädenswil, Switzerland)
• Martin Parniske (Genetics, LMU Munich, Biocenter, Großhaderner Str. 4, 82152 Martinsried, Germany)
• Kirill Demchenko (Laboratory of Plant Anatomy & Morphology, V.L. Komarov Botanical Institute, Russian Academy of Sciences, 2 Prof. Popov Street, St.-Petersburg 197376, St.-Petersburg, Russia)
• Olga Voitsekhovskaja (Department of Plant Physiologial Ecology, V.L. Komarov Botanical Institute, Russian Academy of Sciences, 2 Prof. Popov Street, 197376 St. Petersburg, Russia)
• Daniel Wibberg and Jörn Kalinowski (CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany)
• Rolf Hilker (Department of Bioinformatics and Systems Biology, Justus-Liebig University Giessen, Heinrich-Buff-Ring 58, 35392 Giessen, Germany)
• Anita Sellstedt (Department of Physiological Botany, Umeå University, 901 87 Umeå, Sweden)
• Pooja Jha Maity (Department of Botany, University of Delhi, Delhi 110 007, India)
• Patricia Santos and Dylan Kosma (Department of Biochemistry and Molecular Biology, University of Nevada, Reno 89557, NV, USA)
• Petar Pujic, Nicole Alloisio and Philippe Normand (Ecologie Microbienne, Universite Lyon 1, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France)
• Sabine Zimmermann (Biochimie et Physiologie Moleéculaire des Plantes, UMR 5004 CNRS/INRA/SupAgro/UM2, Campus INRA/SupAgro, 2 Place Viala, 34060 Montpellier Cedex 2, France)
• Ana Ribeiro-Barros (ECO-BIO/IICT, Quinta do Marquês, 2784-505 Oeiras, Portugal)
• Ulrike Mathesius (Division of Plant Science, Research School of Biology, Australian National University, Canberra ACT 2601, Australia)
• Didier Bogusz and Claudine Franche (IRD Montpellier, France)
• Beth Mullin (Department of Botany, University of Tennessee, Knoxville, TN 37996-1100, USA)
• Franziska Krajinski (Institute of Biology, Applied and General Botany Lab, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany)
• Christoph Ziegenhain and Wolfgang Enard (Anthropology and Human Genetics, Department Biology II, Faculty of Biology, Ludwig-Maximilians University Munich, Großhaderner Straße 2, 82152 Martinsried, Germany)
• Marcel Bucher (Botanical Institute, Cologne Biocenter, Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, 50674 Cologne, Germany)

A selection from Stockholm University publication database

• Candidatus Frankia Datiscae Dg1, the Actinobacterial Microsymbiont of Datisca glomerata, Expresses the Canonical nod Genes nodABC in Symbiosis with Its Host Plant

  2015. Tomas Persson (et al.). PLoS ONE 10 (5)

  Article

  Frankia strains are nitrogen-fixing soil actinobacteria that can form root symbioses with actinorhizal plants. Phylogenetically, symbiotic frankiae can be divided into three clusters, and this division also corresponds to host specificity groups. The strains of cluster II which form symbioses with actinorhizal Rosales and Cucurbitales, thus displaying a broad host range, show suprisingly low genetic diversity and to date can not be cultured. The genome of the first representative of this cluster, Candidatus Frankia datiscae Dg1 (Dg1), a microsymbiont of Datisca glomerata, was recently sequenced. A phylogenetic analysis of 50 different housekeeping genes of Dg1 and three published Frankia genomes showed that cluster II is basal among the symbiotic Frankia clusters. Detailed analysis showed that nodules of Datisca glomerata, independent of the origin of the inoculum, contain several closely related cluster II Frankia operational taxonomic units. Actinorhizal plants and legumes both belong to the nitrogen-fixing plant clade, and bacterial signaling in both groups involves the common symbiotic pathway also used by arbuscular mycorrhizal fungi. However, so far, no molecules resembling rhizobial Nod factors could be isolated from Frankia cultures. Alone among Frankia genomes available to date, the genome of Dg1 contains the canonical nod genes nodA, nodB and nodC known from rhizobia, and these genes are arranged in two operons which are expressed in Datisca glomerata nodules. Furthermore, Frankia Dg1 nodC was able to partially complement a Rhizobium leguminosarum A34 nodC::Tn5 mutant. Phylogenetic analysis showed that Dg1 Nod proteins are positioned at the root of both alpha- and beta-rhizobial NodABC proteins. NodA-like acyl transferases were found across the phylum Actinobacteria, but among Proteobacteria only in nodulators. Taken together, our evidence indicates an Actinobacterial origin of rhizobial Nod factors.

  Read more about Candidatus Frankia Datiscae Dg1, the Actinobacterial Microsymbiont of Datisca glomerata, Expresses the Canonical nod Genes nodABC in Symbiosis with Its Host Plant

• Piriformospora indica affects plant growth by auxin production

  2007. Anke Sirrenberg (et al.). Physiologia Plantarum 131, 581–589

  Article

  Piriformospora indica has been shown to improve the growth of many plant species including Arabidopsis thaliana, but the mechanism by which this is achieved is still unclear. Arabidopsis root colonization by P. indica was examined in sterile culture on the medium of Murashige and Skoog. P. indica formed intracellular structures in Arabidopsis root epidermal cells and caused changes in root growth, leading to stunted and highly branched root systems. This effect was because of a diffusible factor and could be mimicked by IAA. In addition, P. indica was shown to produce IAA in liquid culture. We suggest that auxin production affecting root growth is responsible for, or at least contributes to, the beneficial effect of P. indica on its host plants.

  Read more about Piriformospora indica affects plant growth by auxin production

• The diversity of actinorhizal symbiosis

  2012. Katharina Pawlowski, Kirill N. Demchenko. Protoplasma 249 (4), 967-979

  Article

  Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.

  Read more about The diversity of actinorhizal symbiosis

• Jasmonate biosynthesis in legume and actinorhizal nodules

  2011. Katharina Pawlowski (et al.). New Phytologist 189 (2), 568-579

  Article

  • Jasmonic acid (JA) is a plant signalling compound that has been implicated in theregulation of mutualistic symbioses. In order to understand the spatial distributionof JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined thelocalization of allene oxide cyclase (AOC) which catalyses a committed step inJA biosynthesis. In all nodule types analysed, AOC was detected exclusively inuninfected cells.

  • The levels of JA were compared in the roots and nodules of the three plantspecies. The nodules and noninoculated roots of the two actinorhizal species, andthe root systems of M. truncatula, noninoculated or nodulated with wild-type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not showsignificant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance.

  • To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1-sense or MtAOC1-RNAi construct were inoculated with S. meliloti. Neither an increase nor reductionin AOC levels resulted in altered nodule formation.

  • These data suggest that jasmonates are not involved in the development andfunction of root nodules.

  Read more about Jasmonate biosynthesis in legume and actinorhizal nodules

• Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster: They Come in Teams

  2019. Daniel Wibberg (et al.). Genome Biology and Evolution 11 (8), 2273-2291

  Article

  Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to performsymbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that NorthAmerican strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua NewGuinea, represents a novel species, postulated as Candidatus Frankiameridionalis. All Frankia-enrichedmetagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

  Read more about Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster

• A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny

  2020. Defeng Shen (et al.). The Plant Cell 32 (6), 1868-1885

  Article

  A homeotic mutation in Medicago truncatula NODULE ROOT1 converts legume-type nodules into actinorhizal-type nodules, suggesting that the two nodule types have a shared evolutionary origin. Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

  Read more about A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny

• Candidatus Frankia nodulisporulans sp. nov., an Alnus glutinosa-infective Frankia species unable to grow in pure culture and able to sporulate in-planta

  2020. Aude Herrera-Belaroussi (et al.). Systematic and Applied Microbiology 43 (6)

  Article

  We describe a new Frankia species, for three non-isolated strains obtained from Alnus glutinosa in France and Sweden, respectively. These strains can nodulate several Alnus species (A. glutinosa, A. incana, A. alno-betula), they form hyphae, vesicles and sporangia in the root nodule cortex but have resisted all attempts at isolation in pure culture. Their genomes have been sequenced, they are significantly smaller than those of other Alnus-infective species (5 Mb instead of 7.5 Mb) and are very closely related to one another (ANI of 100%). The name Candidatus Frankia nodulisporulans is proposed.

  Read more about Candidatus Frankia nodulisporulans sp. nov., an Alnus glutinosa-infective Frankia species unable to grow in pure culture and able to sporulate in-planta

Show all publications by Katharina Pawlowski at Stockholm University