Root Nodule Symbiosis Research Articles

Molecular Basis of Root Nodule Symbiosis between Bradyrhizobium and 'Crack-Entry' Legume Groundnut (Arachis hypogaea L.).

Nitrogen is one of the essential plant nutrients and a major factor limiting crop productivity. To meet the requirements of sustainable agriculture, there is a need to maximize biological nitrogen fixation in different crop species. Legumes are able to establish root nodule symbiosis (RNS) with nitrogen-fixing soil bacteria which are collectively called rhizobia. This mutualistic association is highly specific, and each rhizobia species/strain interacts with only a specific group of legumes, and vice versa. Nodulation involves multiple phases of interactions ranging from initial bacterial attachment and infection establishment to late nodule development, characterized by a complex molecular signalling between plants and rhizobia. Characteristically, legumes like groundnut display a bacterial invasion strategy popularly known as “crack-entry’’ mechanism, which is reported approximately in 25% of all legumes. This article accommodates critical discussions on the bacterial infection mode, dynamics of nodulation, components of symbiotic signalling pathway, and also the effects of abiotic stresses and phytohormone homeostasis related to the root nodule symbiosis of groundnut and Bradyrhizobium. These parameters can help to understand how groundnut RNS is programmed to recognize and establish symbiotic relationships with rhizobia, adjusting gene expression in response to various regulations. This review further attempts to emphasize the current understanding of advancements regarding RNS research in the groundnut and speculates on prospective improvement possibilities in addition to ways for expanding it to other crops towards achieving sustainable agriculture and overcoming environmental challenges.

Reactive Sulfur Species Interact with Other Signal Molecules in Root Nodule Symbiosis in Lotus japonicus.

Reactive sulfur species (RSS) function as strong antioxidants and are involved in various biological responses in animals and bacteria. Few studies; however, have examined RSS in plants. In the present study, we clarified that RSS are involved in root nodule symbiosis in the model legume Lotus japonicus. Polysulfides, a type of RSS, were detected in the roots by using a sulfane sulfur-specific fluorescent probe, SSP4. Supplying the sulfane sulfur donor Na2S3 to the roots increased the amounts of both polysulfides and hydrogen sulfide (H2S) in the roots and simultaneously decreased the amounts of nitric oxide (NO) and reactive oxygen species (ROS). RSS were also detected in infection threads in the root hairs and in infected cells of nodules. Supplying the sulfane sulfur donor significantly increased the numbers of infection threads and nodules. When nodules were immersed in the sulfane sulfur donor, their nitrogenase activity was significantly reduced, without significant changes in the amounts of NO, ROS, and H2S. These results suggest that polysulfides interact with signal molecules such as NO, ROS, and H2S in root nodule symbiosis in L. japonicus. SSP4 and Na2S3 are useful tools for study of RSS in plants.

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

The Evolutionary Aspects of Legume Nitrogen-Fixing Nodule Symbiosis.

Nitrogen-fixing root nodule symbiosis can sustain the development of the host plants under nitrogen-limiting conditions. Such symbiosis occurs only in a clade of angiosperms known as the nitrogen-fixing clade (NFC). It has long been proposed that root nodule symbiosis evolved several times (in parallel) in the NFC. Two recent phylogenomic studies compared the genomes of nodulating and related non-nodulating species across the four orders of the NFC and found that genes essential for nodule formation are lost or pseudogenized in the non-nodulating species. As these symbiosis genes are specifically involved in the symbiotic interaction, it means that the presence of pseudogenes and the loss of symbiosis genes strongly suggest that their ancestor, which still had functional genes, most likely had a symbiosis with nitrogen-fixing bacteria. These findings agree with the hypothesis that nodulation evolved once at the common ancestor of the NFC, and challenge the hypothesis of parallel evolution. In this chapter, we will cover the current understandings on actinorhizal-type and legume nodule development, and discuss the evolution of the legume nodule type.

Optimization of Hairy Root Transformation for the Functional Genomics in Chickpea: A Platform for Nodule Developmental Studies.

Chickpea is a major protein source in low socio-economic classes and cultivated in marginal soil without fertilizer or irrigation. As a result of its root nodule formation capacity chickpea can directly use atmospheric nitrogen. Chickpea is recalcitrant to stable transformation, particularly root regeneration efficiency of chickpea is low. The composite plant-based system with a non-transformed shoot and transformed root is particularly important for root biologist and this approach has already been used successfully for root nodule symbiosis, arbuscular mycorrhizal symbiosis, and other root-related studies. Use of fluorescent marker-based approach can accurately identify the transformed root from its non-transgenic counterpart. RNAi-based gene knockout, overexpression of genes, promoter GUS analysis to understand tissue specific expression and localization of protein can be achieved using the hairy root-based system. We have already published a hairy root-based transformation and composite plant regeneration protocol of chickpea. Here we are describing the recent modification that we have made to increase the transformation frequency and nodule morphology. Further, we have developed a pouch based artificial system, large number of plants can be scored for its nodule developmental phenotype, by using this system.

CLE-HAR1 Systemic Signaling and NIN-Mediated Local Signaling Suppress the Increased Rhizobial Infection in the daphne Mutant of Lotus japonicus

Legumes survive in nitrogen-limited soil by forming a symbiosis with rhizobial bacteria. During root nodule symbiosis, legumes strictly control the development of their symbiotic organs, the nodules, in a process known as autoregulation of nodulation (AON). The study of hypernodulation mutants has elucidated the molecular basis of AON. Some hypernodulation mutants show an increase in rhizobial infection in addition to developmental alteration. However, the relationship between the AON and the regulation of rhizobial infection has not been clarified. We previously isolated daphne, a nodule inception (nin) allelic mutant, in Lotus japonicus. This mutant displayed dramatically increased rhizobial infection, suggesting the existence of NIN-mediated negative regulation of rhizobial infection. Here, we investigated whether the previously isolated components of AON, especially CLAVATA3/ESR (CLE)-RELATED-ROOT SIGNAL1 (CLE-RS1), CLE-RS2, and their putative receptor HYPERNODULATION AND ABERRANT ROOT FORMATION1 (HAR1), were able to suppress increased infection in the daphne mutant. The constitutive expression of LjCLE-RS1/2 strongly reduced the infection in the daphne mutant in a HAR1-dependent manner. Moreover, reciprocal grafting analysis showed that strong reduction of infection in daphne rootstock constitutively expressing LjCLE-RS1 was canceled by a scion of the har1 or klavier mutant, the genes responsible for encoding putative LjCLE-RS1 receptors. These data indicate that rhizobial infection is also systemically regulated by CLE-HAR1 signaling, a component of AON. In addition, the constitutive expression of NIN in daphne har1 double-mutant roots only partially reduced the rhizobial infection. Our findings indicate that the previously identified NIN-mediated negative regulation of infection involves unknown local signaling, as well as CLE-HAR1 long-distance signaling.

Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume\u2013rhizobia symbiosis

Root nodule (RN) symbiosis is a mutualistic interaction observed between nitrogen-fixing soil bacteria and nodulating plants, which are scattered in only four orders of angiosperms called nitrogen-fixing clade. Most of legumes engage in RN symbiosis with rhizobia. Molecular genetic analyses with legumes and non-leguminous nodulating plants revealed that RN symbiosis utilizes early signalling components that are required for symbiosis with arbuscular mycorrhizal (AM) fungi. However detailed evolutionary processes are still largely unknown. Comparative analyses with non-nodulating species phylogenetically related to legumes could be better strategies to study the evolution of RN symbiosis in legumes. Polygala paniculata is a non-leguminous species that belongs to a family different from legumes but that is classified into the same order, Fabales. It has appropriate characteristics for cultivation in laboratories: small body size, high fertility and short lifecycles. Therefore, we further assessed whether this species is suitable as a model species for comparative studies with legumes. We first validated that the plant we obtained in Palau was truly P. paniculata by molecular phylogenetic analysis using rbcL sequences. The estimated genome size of this species was less than those of two model legumes, Lotus japonicus and Medicago truncatula. We determined conditions for cultivation in vitro and for hairy root formation from P. paniculata seedlings. It would facilitate to investigate gene functions in this species. The ability of P. paniculata to interact with AM fungi was confirmed by inoculation with Rhizophagus irregularis, suggesting the presence of early signalling factors that might be involved in RN symbiosis. Unexpectedly, branching of root hairs was observed when inoculated with Mesorhizobium loti broad host range strain NZP2037, indicating that P. paniculata has the biological potential to respond to rhizobia. We propose that P. paniculata is used as a model plant for the evolutionary study of RN symbiosis.

LCO Receptors Involved in Arbuscular Mycorrhiza Are Functional for Rhizobia Perception in Legumes.

SummaryBacterial lipo-chitooligosaccharides (LCOs) are key mediators of the nitrogen-fixing root nodule symbiosis (RNS) in legumes. The isolation of LCOs from arbuscular mycorrhizal fungi suggested that LCOs are also signaling molecules in arbuscular mycorrhiza (AM). However, the corresponding plant receptors have remained uncharacterized. Here we show that petunia and tomato mutants in the LysM receptor-like kinases LYK10 are impaired in AM formation. Petunia and tomato LYK10 proteins have a high affinity for LCOs (Kd in the nM range) comparable to that previously reported for a legume LCO receptor essential for the RNS. Interestingly, the tomato and petunia LYK10 promoters, when introduced into a legume, were active in nodules similarly to the promoter of the legume orthologous gene. Moreover, tomato and petunia LYK10 coding sequences restored nodulation in legumes mutated in their orthologs. This combination of genetic and biochemical data clearly pinpoints Solanaceous LYK10 as part of an ancestral LCO perception system involved in AM establishment, which has been directly recruited during evolution of the RNS in legumes.

A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus.

Legumes develop root nodules in symbiosis with nitrogen-fixing rhizobial bacteria. Rhizobia evoke cell division of differentiated cortical cells into root nodule primordia for accommodating bacterial symbionts. In this study, we show that NODULE INCEPTION (NIN), a transcription factor in Lotus japonicus that is essential for initiating cortical cell divisions during nodulation, regulates the gene ASYMMETRIC LEAVES 2-LIKE 18/LATERAL ORGAN BOUNDARIES DOMAIN 16a (ASL18/LBD16a). Orthologs of ASL18/LBD16a in nonlegume plants are required for lateral root development. Coexpression of ASL18a and the CCAAT box-binding protein Nuclear Factor-Y (NF-Y) subunits, which are also directly targeted by NIN, partially suppressed the nodulation-defective phenotype of L. japonicus daphne mutants, in which cortical expression of NIN was attenuated. Our results demonstrate that ASL18a and NF-Y together regulate nodule organogenesis. Thus, a lateral root developmental pathway is incorporated downstream of NIN to drive nodule symbiosis.

Transcriptome analysis of the differential effect of the NADPH oxidase gene RbohB in Phaseolus vulgaris roots following Rhizobium tropici and Rhizophagus irregularis inoculation

BackgroundReactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. Rboh is a multigene family comprising nine members (RbohA–I) in common bean (Phaseolus vulgaris). The RNA interference-mediated silencing of RbohB (PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, the PvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed that PvRbohB positively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization by Rhizophagus irregularis in P. vulgaris.ResultsTo corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots.ConclusionsOur research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P. vulgaris, as well as the differential roles that RbohB plays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.

Glutathione is Involved in Detoxification of Peroxide and Root Nodule Symbiosis of Mesorhizobium huakuii.

Legumes interact with symbiotic rhizobia to produce nitrogen-fixation root nodules under nitrogen-limiting conditions. The contribution of glutathione (GSH) to this symbiosis and anti-oxidative damage was investigated using the M. huakuii gshB (encoding GSH synthetase) mutant. The gshB mutant grew poorly with different monosaccharides, including glucose, sucrose, fructose, maltose, or mannitol, as sole sources of carbon. The antioxidative capacity of gshB mutant was significantly decreased by these treatments with H2O2 under the lower concentrations and cumene hydroperoxide (CUOOH) under the higher concentrations, indicating that GSH plays different roles in response to organic peroxide and inorganic peroxide. The gshB mutant strain displayed no difference in catalase activity, but significantly lower levels of the peroxidase activity and the glutathione reductase activity than the wild type. The same level of catalase activity could be associated with upregulation of the transcriptional activity of the catalase genes under H2O2-induced conditions. The nodules infected by the gshB mutant were severely impaired in abnormal nodules, and showed a nodulation phenotype coupled to a 60% reduction in the nitrogen fixation capacity. A 20-fold decrease in the expression of two nitrogenase genes, nifH and nifD, is observed in the nodules induced by gshB mutant strain. The symbiotic deficiencies were linked to bacteroid early senescence.

A Stable Genetic Transformation System and Implications of the Type IV Restriction System in the Nitrogen-Fixing Plant Endosymbiont Frankia alni ACN14a.

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes that are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. The corresponding endosymbionts, Frankia and the rhizobia, however, are distantly related groups of bacteria, leading to questions about their symbiotic mechanisms and evolutionary history. To date, a stable system of electrotransformation has been lacking in Frankia despite numerous attempts by research groups worldwide. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation. Here we report the successful electrotransformation of the model strain F. alni ACN14a with an unmethylated, broad host-range replicating plasmid, expressing chloramphenicol-resistance for selection and GFP as a marker of gene expression. This system circumvented the type IV restriction barrier and allowed the stable maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the expression of egfp under the control of the nif gene cluster promoter was localized using fluorescence imaging, the expression of nitrogen fixation in nitrogen-limited culture was localized in Frankia vesicles but not in hyphae. The ability to separate gene expression patterns between Frankia hyphae and vesicles will enable deeper comparisons of molecular signaling and metabolic exchange between Frankia-actinorhizal and rhizobia-legume symbioses to be made, and may broaden potential applications in agriculture. Further downstream applications are possible, including gene knock-outs and complementation, to open up a range of experiments in Frankia and its symbioses. Additionally, in the transcriptome of F. alni ACN14a, type IV restriction enzymes were highly expressed in nitrogen-replete culture but their expression strongly decreased during symbiosis. The down-regulation of type IV restriction enzymes in symbiosis suggests that horizontal gene transfer may occur more frequently inside the nodule, with possible new implications for the evolution of Frankia.

The Rpf84 gene, encoding a ribosomal large subunit protein, RPL22, regulates symbiotic nodulation in Robinia pseudoacacia.

A homologue of the ribosomal protein L22e, Rpf84, regulates root nodule symbiosis by mediating the infection process of rhizobia and preventing bacteroids from degradation in Robinia pseudoacacia. Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.

ERN1 and CYCLOPS coordinately activate NIN signaling to promote infection thread formation in Lotus japonicus

Legumes engage in symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia, under nitrogen-limited conditions. In many legumes, the root invasion of rhizobia is mediated by infection threads (ITs), tubular invaginations of the host cell wall and plasma membrane, developed from infection foci of deformed root hairs. IT formation is regulated by a series of signal transduction in host root. Nodulation signals activate the host transcription factor (TF), CYCLOPS, which directly induces expression of two TF genes, ERF REQUIRED FOR NODULATION1 (ERN1) and NODULE INCEPTION (NIN), essential for IT development. Here, we explored the relationship among these three symbiotic TF genes in the model legume Lotus japonicus and examined how their interplay contributes to IT formation. qRT-PCR analysis showed that NIN expression induced by rhizobial infection was attenuated in ern1-1, and further declined in cyclops-3 ern1-1. ERN1 overexpression led to induction of NIN expression in cyclops-3 ern1-1 in the presence of rhizobia. Thus, in addition to CYCLOPS, ERN1 is able to increase the NIN expression level depending on infection. Furthermore, consistent with this transcriptional hierarchy, ectopic expression of ERN1 as well as NIN suppressed the IT-deficient cyclops-3 phenotype, but ERN1 failed to confer ITs in the nin-2 root. However, the ern1-1 symbiotic epidermal phenotype was not suppressed by the NIN ectopic expression. The cyclops-3 ern1-1 double mutant was less sensitive to rhizobial infection than the single mutants and defective in the symbiotic root hair response at earlier stages. This more severe phenotype of the double mutant suggests a role for ERN1 that independent of the CYCLOPS-mediated transcriptional regulation. We conclude that ERN1 is involved in regulating NIN expression in addition to CYCLOPS, and these TFs coordinately promote the symbiotic root hair response and IT development. Our data help to reveal the extensive role of ERN1 in root nodule symbiosis signaling.

Salt-stress secondary metabolite signatures involved in the ability of Casuarina glauca to mitigate oxidative stress

The model actinorhizal plant Casuarina glauca is characterized by its capacity to establish nitrogen-fixing root-nodule symbiosis with Frankia bacteria and by its resilience to thrive under extreme environments, such as high salinity. Previous studies have shown that salt tolerance in C. glauca is linked to photosynthetic, primary metabolic adjustments and to an effective antioxidant machinery. This study aimed at analyzing the secondary metabolite composition of tissues from nodulated (NOD+) and non-nodulated (KNO3+) C. glauca plants (nodules, roots, and branchlets) with liquid-chromatography-high resolution mass spectrometry (LC-HRMS) to evaluate the effect of three NaCl concentrations (200, 400 and 600 mM) on the secondary metabolite composition of these two plant groups. In the branchlets, significant accumulation of almost all annotated secondary metabolites, including flavonoids and their glycosides, was mainly induced by the highest salt concentration. Furthermore, the different secondary metabolite levels between roots and nodules were associated to the previously reported impairment of symbiotic activity from 200 mM NaCl onwards. Ionome analysis in C. glauca branchlets revealed salt stress-induced changes in ion homeostasis, which in turn, affected the K+/Na+ ratio. Nevertheless, despite these changes C. glauca plants can still maintain its primary metabolic activity. Moreover, our results suggest that C. glauca plants activate a flavonoid-based secondary antioxidant system to mitigate the impacts of oxidative stress, thereby enhancing their salt stress tolerance.

Ectopic or Over-Expression of Class 1 Phytoglobin Genes Confers Flooding Tolerance to the Root Nodules of Lotus japonicus by Scavenging Nitric Oxide.

Flooding limits biomass production in agriculture. Leguminous plants, important agricultural crops, use atmospheric dinitrogen gas as nitrogen nutrition by symbiotic nitrogen fixation with rhizobia, but this root-nodule symbiosis is sometimes broken down by flooding of the root system. In this study, we analyzed the effect of flooding on the symbiotic system of transgenic Lotus japonicus lines which overexpressed class 1 phytoglobin (Glb1) of L. japonicus (LjGlb1-1) or ectopically expressed that of Alnus firma (AfGlb1). In the roots of wild-type plants, flooding increased nitric oxide (NO) level and expression of senescence-related genes and decreased nitrogenase activity; in the roots of transgenic lines, these effects were absent or less pronounced. The decrease of chlorophyll content in leaves and the increase of reactive oxygen species (ROS) in roots and leaves caused by flooding were also suppressed in these lines. These results suggest that increased levels of Glb1 help maintain nodule symbiosis under flooding by scavenging NO and controlling ROS.

A protein complex required for polar growth of rhizobial infection threads

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads.

Evolution of Angiosperm Pollen. 7. Nitrogen-fixing Clade

Nitrogen-fixing symbiosis in root nodules is known in only 10 families, which are distributed among a clade of four orders and delimited as the nitrogen-fixing clade. As the seventh in a series that examines pollen morphological distribution and evolution in the angiosperms, this paper focuses on pollen morphological character states of the nitrogen-fixing clade. To illustrate the palynological diversity of the clade, we first examined pollen grains from 26 species with light electron, scanning electron, and transmission electron microscopy. Second, we used a reduced data matrix from Li et al. (2015) to reconstruct a maximum likelihood tree and then optimized 18 pollen character states onto the tree using Fitch parsimony, maximum likelihood, and hierarchical Bayesian inference. Finally, 12 plesiomorphic states for the nitrogen-fixing clade were inferred unambiguously under all methods, and more than 40 clades (or lineages) at or above familial level were characterized by unambiguous pollen character state changes in at least one of the optimizations. We found a number of evolutionary trends for changes in pollen character states. These include increasing grain size, increasing aperture number accompanied by concomitant changes in aperture position (from equatorial to global) and aperture shape (from colpate to colporate), and increasing complexity of tectum ornamentation. There was a strong correlation between some pollen characters (prolate shape class, lobe outline in polar view, colpate ectoaperture, lalongate and lolongate endoaperture, absent supratectal element, reticulate tectum) and insect pollination, while other pollen characters—simple aperture structure, porate ectoaperture, circular endoaperture, present and gemmate or echinate supratectal element, and imperforate tectum—were strongly correlated with wind pollination. In addition, rugulate tectum was significantly correlated with shrub habit while larger pollen size was significantly correlated with vine habit; the helophytic habitat was significantly correlated with having two apertures. Our study provides rich evidence for the phylogenetic significance of pollen morphological diversity in the nitrogen-fixing clade.

Dryas as a Model for Studying the Root Symbioses of the Rosaceae.

The nitrogen-fixing root nodule symbiosis is restricted to four plant orders: Fabales (legumes), Fagales, Cucurbitales and Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae). Interestingly all of the Rosaceae genera confirmed to contain nodulating species (i.e., Cercocarpus, Chamaebatia, Dryas, and Purshia) belong to a single subfamily, the Dryadoideae. The Dryas genus is particularly interesting from an evolutionary perspective because it contains closely related nodulating (Dryas drummondii) and non-nodulating species (Dryas octopetala). The close phylogenetic relationship between these two species makes Dryas an ideal model genus to study the genetic basis of nodulation by whole genome comparison and classical genetics. Therefore, we established methods for plant cultivation, transformation and DNA extraction for these species. We optimized seed surface sterilization and germination methods and tested growth protocols ranging from pots and Petri dishes to a hydroponic system. Transgenic hairy roots were obtained by adapting Agrobacterium rhizogenes-based transformation protocols for Dryas species. We compared several DNA extraction protocols for their suitability for subsequent molecular biological analysis. Using CTAB extraction, reproducible PCRs could be performed, but CsCl gradient purification was essential to obtain DNA in sufficient purity for high quality de novo genome sequencing of both Dryas species. Altogether, we established a basic toolkit for the culture, transient transformation and genetic analysis of Dryas sp.

Symbiotic Performance of Sinorhizobium meliloti Lacking ppGpp Depends on the Medicago Host Species.

Host specificity in the root-nodule symbiosis between legumes and rhizobia is crucial for the establishment of a successful interaction and ammonia provision to the plant. The specificity is mediated by plant-bacterial signal exchange during early stages of interaction. We observed that a Sinorhizobium meliloti mutant ∆relA, which is deficient in initiating the bacterial stringent response, fails to nodulate Medicago sativa (alfalfa) but successfully infects Medicago truncatula. We used biochemical, histological, transcriptomic, and imaging approaches to compare the behavior of the S. meliloti ∆relA mutant and wild type (WT) on the two plant hosts. ∆relA performed almost WT-like on M. truncatula, except for reduced nitrogen-fixation capacity and a disorganized positioning of bacteroids within nodule cells. In contrast, ∆relA showed impaired root colonization on alfalfa and failed to infect nodule primordia. Global transcriptome analyses of ∆relA cells treated with the alfalfa flavonoid luteolin and of mature nodules induced by the mutant on M. truncatula revealed normal nod gene expression but overexpression of exopolysaccharide biosynthesis genes and a slight suppression of plant defense-like reactions. Many RelA-dependent transcripts overlap with the hypo-osmolarity-related FeuP regulon or are characteristic of stress responses. Based on our findings, we suggest that RelA is not essential until the late stages of symbiosis with M. truncatula, in which it may be involved in processes that optimize nitrogen fixation.