A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation.

BACKGROUND: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. RESULTS: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. CONCLUSION: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

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

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.

The BOP-type co-transcriptional regulator NODULE ROOT1 promotes stem secondary growth of the tropical Cannabaceae tree Parasponia andersonii.

Tree stems undergo a massive secondary growth in which secondary xylem and phloem tissues arise from the vascular cambium. Vascular cambium activity is driven by endogenous developmental signalling cues and environmental stimuli. Current knowledge regarding the genetic regulation of cambium activity and secondary growth is still far from complete. The tropical Cannabaceae tree Parasponia andersonii is a non-legume research model of nitrogen-fixing root nodulation. Parasponia andersonii can be transformed efficiently, making it amenable for CRISPR-Cas9-mediated reverse genetics. We considered whether P. andersonii also could be used as a complementary research system to investigate tree-related traits, including secondary growth. We established a developmental map of stem secondary growth in P. andersonii plantlets. Subsequently, we showed that the expression of the co-transcriptional regulator PanNODULE ROOT1 (PanNOOT1) is essential for controlling this process. PanNOOT1 is orthologous to Arabidopsis thaliana BLADE-ON-PETIOLE1 (AtBOP1) and AtBOP2, which are involved in the meristem-to-organ-boundary maintenance. Moreover, in species forming nitrogen-fixing root nodules, NOOT1 is known to function as a key nodule identity gene. Parasponia andersonii CRISPR-Cas9 loss-of-function Pannoot1 mutants are altered in the development of the xylem and phloem tissues without apparent disturbance of the cambium organization and size. Transcriptomic analysis showed that the expression of key secondary growth-related genes is significantly down-regulated in Pannoot1 mutants. This allows us to conclude that PanNOOT1 positively contributes to the regulation of stem secondary growth. Our work also demonstrates that P. andersonii can serve as a tree research system.

High Salt Levels Reduced Dissimilarities in Root-Associated Microbiomes of Two Barley Genotypes.

Plants harbor in and at their roots bacterial microbiomes that contribute to their health and fitness. The microbiome composition is controlled by the environment and plant genotype. Previously, it was shown that the plant genotype-dependent dissimilarity of root microbiome composition of different species becomes smaller under drought stress. However, it remains unknown whether this reduced plant genotype-dependent effect is a specific response to drought stress or a more generic response to abiotic stress. To test this, we studied the effect of salt stress on two distinct barley (Hordeum vulgare L.) genotypes: the reference cultivar Golden Promise and the Algerian landrace AB. As inoculum, we used soil from salinized and degraded farmland on which barley was cultivated. Controlled laboratory experiments showed that plants inoculated with this soil displayed growth stimulation under high salt stress (200 mM) in a plant genotype-independent manner, whereas the landrace AB also showed significant growth stimulation at low salt concentrations. Subsequent analysis of the root microbiomes revealed a reduced dissimilarity of the bacterial communities of the two barley genotypes in response to high salt, especially in the endophytic compartment. High salt level did not reduce α-diversity (richness) in the endophytic compartment of both plant genotypes but was associated with an increased number of shared strains that respond positively to high salt. Among these, Pseudomonas spp. were most abundant. These findings suggest that the plant genotype-dependent microbiome composition is altered generically by abiotic stress.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pseudogenization of the rhizobium-responsive EXOPOLYSACCHARIDE RECEPTOR in Parasponia is a rare event in nodulating plants.

BACKGROUND: Nodule symbiosis with diazotrophic Frankia or rhizobium occurs in plant species belonging to ten taxonomic lineages within the related orders Fabales, Fagales, Cucurbitales, and Rosales. Phylogenomic studies indicate that this nitrogen-fixing nodulation trait has a single evolutionary origin. In legume model plants, the molecular interaction between plant and rhizobium microsymbiont is mapped to a significant degree. A specific LysM-type receptor kinase, LjEPR3 in Lotus japonicus and MtLYK10 in Medicago truncatula, was found to act in a secondary identity-based mechanism, controlling intracellular rhizobium infection. Furthermore, LjEPR3 showed to bind surface exopolysaccharides of Mesorhizobium loti, the diazotrophic microsymbiont of L. japonicus. EPR3 orthologous genes are not unique to legumes. Surprisingly, however, its ortholog EXOPOLYSACCHARIDE RECEPTOR (EPR) is pseudogenized in Parasponia, the only lineage of non-legume plants that nodulate also with rhizobium. RESULTS: Analysis of genome sequences showed that EPR3 orthologous genes are highly conserved in nodulating plants. We identified a conserved retrotransposon insertion in the EPR promoter region in three Parasponia species, which associates with defected transcriptional regulation of this gene. Subsequently, we studied the EPR gene of two Trema species as they represent the sister genus of Parasponia for which it is assumed it lost the nitrogen-fixing nodulation trait. Both Trema species possess apparently functional EPR genes that have a nodulation-specific expression profile when introduced into a Parasponia background. This indicates the EPR gene functioned in nodulation in the Parasponia-Trema ancestor. CONCLUSION: We conclude that nodule-specific expression of EPR3 orthologous genes is shared between the legume and Parasponia-Trema lineage, suggesting an ancestral function in the nitrogen-fixing nodulation trait. Pseudogenization of EPR in Parasponia is an exceptional case in nodulating plants. We speculate that this may have been instrumental to the microsymbiont switch -from Frankia to rhizobium- that has occurred in the Parasponia lineage and the evolution of a novel crack entry infection mechanism.

A Remote cis-Regulatory Region Is Required for NIN Expression in the Pericycle to Initiate Nodule Primordium Formation in Medicago truncatula.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Evolution of endemism on a young tropical mountain.

Tropical mountains are hot spots of biodiversity and endemism, but the evolutionary origins of their unique biotas are poorly understood. In varying degrees, local and regional extinction, long-distance colonization, and local recruitment may all contribute to the exceptional character of these communities. Also, it is debated whether mountain endemics mostly originate from local lowland taxa, or from lineages that reach the mountain by long-range dispersal from cool localities elsewhere. Here we investigate the evolutionary routes to endemism by sampling an entire tropical mountain biota on the 4,095-metre-high Mount Kinabalu in Sabah, East Malaysia. We discover that most of its unique biodiversity is younger than the mountain itself (6 million years), and comprises a mix of immigrant pre-adapted lineages and descendants from local lowland ancestors, although substantial shifts from lower to higher vegetation zones in this latter group were rare. These insights could improve forecasts of the likelihood of extinction and 'evolutionary rescue' in montane biodiversity hot spots under climate change scenarios.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

GeneNoteBook, a collaborative notebook for comparative genomics.

SUMMARY: Analysis and comparison of genomic and transcriptomic datasets have become standard procedures in biological research. However, for non-model organisms no efficient tools exist to visually work with multiple genomes and their metadata, and to annotate such data in a collaborative way. Here we present GeneNoteBook: a web based collaborative notebook for comparative genomics. GeneNoteBook allows experimental and computational researchers to query, browse, visualize and curate bioinformatic analysis results for multiple genomes. GeneNoteBook is particularly suitable for the analysis of non-model organisms, as it allows for comparing newly sequenced genomes to those of model organisms. AVAILABILITY AND IMPLEMENTATION: GeneNoteBook is implemented as a node.js web application and depends on MongoDB and NCBI BLAST. Source code is available at https://github.com/genenotebook/genenotebook. Additionally, GeneNoteBook can be installed through Bioconda and as a Docker image. Full installation instructions and online documentation are available at https://genenotebook.github.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF-YA1 transcription factors as a core genetic network in nitrogen-fixing nodule symbioses.

●Nitrogen-fixing nodulation occurs in 10 taxonomic lineages, with either rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume-rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF-Y) complex. ●It is hypothesized that nodulation has a single evolutionary origin c. 110 Ma, followed by many independent losses. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF-YA genes in rhizobium nodulation in a nonlegume system. ●Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are coexpressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. ●This demonstrates that NIN and NF-YA1 have conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis.

The Medicago truncatula nodule identity gene MtNOOT1 is required for coordinated apical-basal development of the root.

BACKGROUND: Legumes can utilize atmospheric nitrogen by hosting nitrogen-fixing bacteria in special lateral root organs, called nodules. Legume nodules have a unique ontology, despite similarities in the gene networks controlling nodule and lateral root development. It has been shown that Medicago truncatula NODULE ROOT1 (MtNOOT1) is required for the maintenance of nodule identity, preventing the conversion to lateral root development. MtNOOT1 and its orthologs in other plant species -collectively called the NOOT-BOP-COCH-LIKE (NBCL) family- specify boundary formation in various aerial organs. However, MtNOOT1 is not only expressed in nodules and aerial organs, but also in developing roots, where its function remains elusive. RESULTS: We show that Mtnoot1 mutant seedlings display accelerated root elongation due to an enlarged root apical meristem. Also, Mtnoot1 mutant roots are thinner than wild-type and are delayed in xylem cell differentiation. We provide molecular evidence that the affected spatial development of Mtnoot1 mutant roots correlates with delayed induction of genes involved in xylem cell differentiation. This coincides with a basipetal shift of the root zone that is susceptible to rhizobium-secreted symbiotic signal molecules. CONCLUSIONS: Our data show that MtNOOT1 regulates the size of the root apical meristem and vascular differentiation. Our data demonstrate that MtNOOT1 not only functions as a homeotic gene in nodule development but also coordinates the spatial development of the root.

A genetically and functionally diverse group of non-diazotrophic Bradyrhizobium spp. colonizes the root endophytic compartment of Arabidopsis thaliana.

BACKGROUND: Diazotrophic Bradyrhizobium spp. are well known for their ability to trigger nodule formation on a variety of legume species. In nodules, Bradyrhizobium utilizes plant-derived carbohydrates in exchange for fixed nitrogen. The genes essential for the nodulation and nitrogen-fixation trait are clustered in a genomic region, which is known as the 'symbiotic island'. Recently, novel non-diazotrophic Bradyrhizobium spp. have been found to be highly abundant in soils, suggesting that these species can also have a 'free-living' life history. However, whether non-diazotrophic Bradyrhizobium spp. can live in association with plants remains elusive. RESULTS: In this study, we show that Bradyrhizobium spp. are common root endophytes of non-legume plant species - including Arabidopsis thaliana (Arabidopsis) - grown in an ecological setting. From a single Arabidopsis root, four Bradyrhizobium sp. strains (designated MOS001 to MOS004) were isolated. Comparative genome analysis revealed that these strains were genetically and functionally highly diverse, but did not harbour the nodulation and the nitrogen fixation gene clusters. Comparative colonization experiments, with MOS strains and nitrogen-fixing symbiotic strains, revealed that all tested Bradyrhizobium spp. can colonize the root endophytic compartment of Arabidopsis. CONCLUSION: This study provides evidence that both diazotrophic and non-diazotrophic Bradyrhizobium spp. colonize the root endophytic compartment of a wide variety of plant species, including the model species Arabidopsis. This demonstrates that plant roots form a major ecological niche for Bradyrhizobium spp., which might be ancestral to the evolution of the nodulation and nitrogen-fixation trait in this genus.

Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity.

Many actinobacteria live in close association with eukaryotes such as fungi, insects, animals and plants. Plant-associated actinobacteria display (endo)symbiotic, saprophytic or pathogenic life styles, and can make up a substantial part of the endophytic community. Here, we characterised endophytic actinobacteria isolated from root tissue of Arabidopsis thaliana (Arabidopsis) plants grown in soil from a natural ecosystem. Many of these actinobacteria belong to the family of Streptomycetaceae with Streptomyces olivochromogenes and Streptomyces clavifer as well represented species. When seeds of Arabidopsis were inoculated with spores of Streptomyces strain coa1, which shows high similarity to S. olivochromogenes, roots were colonised intercellularly and, unexpectedly, also intracellularly. Subsequent exposure of endophytic isolates to plant hormones typically found in root and shoot tissues of Arabidopsis led to altered antibiotic production against Escherichia coli and Bacillus subtilis. Taken together, our work reveals remarkable colonization patterns of endophytic streptomycetes with specific traits that may allow a competitive advantage inside root tissue.

The Medicago genome provides insight into the evolution of rhizobial symbioses.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus.

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.

Quantitative modelling of legume root nodule primordium induction by a diffusive signal of epidermal origin that inhibits auxin efflux.

BACKGROUND: Rhizobium nitrogen fixation in legumes takes place in specialized organs called root nodules. The initiation of these symbiotic organs has two important components. First, symbiotic rhizobium bacteria are recognized at the epidermis through specific bacterially secreted lipo-chitooligosaccharides (LCOs). Second, signaling processes culminate in the formation of a local auxin maximum marking the site of cell divisions. Both processes are spatially separated. This separation is most pronounced in legumes forming indeterminate nodules, such as model organism Medicago truncatula, in which the nodule primordium is formed from pericycle to most inner cortical cell layers. RESULTS: We used computer simulations of a simplified root of a legume that can form indeterminate nodules. A diffusive signal that inhibits auxin transport is produced in the epidermis, the site of rhizobium contact. In our model, all cells have the same response characteristics to the diffusive signal. Nevertheless, we observed the fastest and strongest auxin accumulation in the pericycle and inner cortex. The location of these auxin maxima correlates with the first dividing cells of future nodule primordia in M. truncatula. The model also predicts a transient reduction of the vascular auxin concentration rootward of the induction site as is experimentally observed. We use our model to investigate how competition for the vascular auxin source could contribute to the regulation of nodule number and spacing. CONCLUSION: Our simulations show that the diffusive signal may invoke the strongest auxin accumulation response in the inner root layers, although the signal itself is strongest close to its production site.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

BACKGROUND: Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. RESULTS: Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. CONCLUSIONS: Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.

The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis.

BACKGROUND: Strigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis. RESULTS: We provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2. CONCLUSION: Our data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.

Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.