Medicago Truncatula Nodules Research Articles

Impact of Hypoxia on Nodulation and Growth of the Legume Plant Medicago truncatula in Symbiosis with Sinorhizobium meliloti

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to develop nitrogen-fixing root nodules under nitrogen-limiting conditions. The present study aimed at analysing the impact of two- and four-week long periods of hypoxia on the nodulation and the growth of the legume plant Medicago truncatula (var Jemalong A17) in symbiosis with Sinorhizobium meliloti. Results showed that the nodulation was not affected by moderate hypoxia (4.5% O2), but the number of nodules decreases for high hypoxia (0.1% O2) and anoxia. The analysis of growth parameters (fresh and dry weight, leaf area and thickness, chlorophyll contents) and of various metabolite contents (total sugars, chlorophylls, inorganic nitrogen compounds) in the leaves, roots and nodules showed that nodulated Medicago truncatula tolerates periods of 4 weeks hypoxia and may be considered as a hypoxia-tolerant legume plant.

Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula.

The legume-Rhizobium symbiosis leads to the formation of a new organ, the root nodule, involving coordinated and massive induction of specific genes. Several genes controlling DNA methylation are spatially regulated within the Medicago truncatula nodule, notably the demethylase gene, DEMETER (DME), which is mostly expressed in the differentiation zone. Here, we show that MtDME is essential for nodule development and regulates the expression of 1,425 genes, some of which are critical for plant and bacterial cell differentiation. Bisulphite sequencing coupled to genomic capture enabled the identification of 474 regions that are differentially methylated during nodule development, including nodule-specific cysteine-rich peptide genes. Decreasing DME expression by RNA interference led to hypermethylation and concomitant downregulation of 400 genes, most of them associated with nodule differentiation. Massive reprogramming of gene expression through DNA demethylation is a new epigenetic mechanism controlling a key stage of indeterminate nodule organogenesis during symbiotic interactions.

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula.

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA−). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1–MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.

NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula.

The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops.

Plants are able to lose organs selectively through a process called abscission. This process relies on the differentiation of specialized territories at the junction between organs and the plant body that are called abscission zones (AZ). Several genes control the formation or functioning of these AZ. We have characterized BLADE-ON-PETIOLE (BOP) orthologues from several legume plants and studied their roles in the abscission process using a mutant approach. Here, we show that the Medicago truncatula NODULE ROOT (NOOT), the Pisum sativum COCHLEATA (COCH) and their orthologue in Lotus japonicus are strictly necessary for the abscission of not only petals, but also leaflets, leaves and fruits. We also showed that the expression pattern of the M.truncatula pNOOT::GUS fusion is associated with functional and vestigial AZs when expressed in Arabidopsis. In addition, we show that the stip mutant from Lupinus angustifolius, defective in stipule formation and leaf abscission, is mutated in a BOP orthologue. In conclusion, this study shows that this clade of proteins plays an important conserved role in promoting abscission of all aerial organs studied so far.

Does SUNN-SYMRK Crosstalk occur in Medicago truncatula for regulating nodule organogenesis?

Recently we reported that overexpression of intracellular kinase domain of Symbiosis Receptor Kinase (SYMRK-kd) hyperactivated spontaneous nodulation in Medicago truncatula indicating the importance of SYMRK ectodomain in restricting nodule number. To clarify whether sunn and sickle pathways were overcome by SYMRK-kd for hyperactivation of nodule organogenesis, we overexpressed SYMRK-kd in these mutants and analyzed for spontaneous nodulation in absence of rhizobia. Spontaneous nodulation in skl/SYMRK-kd roots was 2-fold higher than A17/SYMRK-kd roots indicating nodule organogenesis induced by SYMRK-kd to be ethylene sensitive. Intriguingly, sunn/SYMRK-kd roots failed to generate any spontaneous nodule which directly indicate the LRR-RLK SUNN to have a role in SYMRK-kd mediated nodule development under non-symbiotic conditions. We hypothesize a crosstalk between SUNN and SYMRK receptors for activation as well as restriction of nodule development.

Editorial: Plant responses to bacterial quorum sensing molecules.

Editorial: Plant responses to bacterial quorum sensing molecules.

Multiple Autoregulation of Nodulation (AON) Signals Identified through Split Root Analysis of Medicago truncatula sunn and rdn1 Mutants

Nodulation is energetically costly to the host: legumes balance the nitrogen demand with the energy expense by limiting the number of nodules through long-distance signaling. A split root system was used to investigate systemic autoregulation of nodulation (AON) in Medicago truncatula and the role of the AON genes RDN1 and SUNN in the regulatory circuit. Developing nodule primordia did not trigger AON in plants carrying mutations in RDN1 and SUNN genes, while wild type plants had fully induced AON within three days. However, despite lacking an early suppression response, AON mutants suppressed nodulation when roots were inoculated 10 days or more apart, correlated with the maturation of nitrogen fixing nodules. In addition to correlation between nitrogen fixation and suppression of nodulation, suppression by extreme nutrient stress was also observed in all genotypes and may be a component of the observed response due to the conditions of the assay. These results suggest there is more than one systemic regulatory circuit controlling nodulation in M. truncatula. While both signals are present in wild type plants, the second signal can only be observed in plants lacking the early repression (AON mutants). RDN1 and SUNN are not essential for response to the later signal.

Quantification of the Volume and Surface Area of Symbiosomes and Vacuoles of Infected Cells in Root Nodules of Medicago truncatula

Legumes are able to form endosymbiotic interactions with nitrogen-fixing rhizobia. Endosymbiosis takes shape in formation of a symbiotic organ, the root nodule. Medicago truncatula (M. truncatula) nodules contain several zones representing subsequent stages of development. The apical part of the nodule consists of the meristem and the infection zone. At this site, bacteria are released into the host cell from infection threads. Upon release, bacteria are surrounded by a host cell–derived membrane to form symbiosomes. After release, rhizobia grow, divide, and gradually colonize the entire host cell of the fixation zone of root nodules. Therefore, mature infected cells contain thousands of symbiosomes, which remain as individual units among other organelles. Visualization of the organization and dynamics of the symbiosomes as well as other organelles in infected cells of nodules is essential to understand mechanisms regulating the development of endosymbiosis between plants and rhizobia. To examine this highly dynamic developmental process we designed a useful imaging technique that is based on confocal scanning microscopy combined with different fluorescent dyes and GFP-tagged proteins (Gavrin et al., 2014). Here, we describe a protocol for microscopic observation, 3D rendering, and volume/area measurements of symbiosomes and other organelles in infected cells of M. truncatula root nodules. This protocol can be applied for monitoring the development of different host-microbe interactions whether symbiotic or pathogenic.

Root developmental programs shape the Medicago truncatula nodule meristem.

Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation.

Nod factor receptors form heteromeric complexes and are essential for intracellular infection in medicago nodules.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.

Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago.

In legume-rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria.

Involvement of papain and legumain proteinase in the senescence process of Medicago truncatula nodules

The symbiotic interaction between legumes and Rhizobiaceae leads to the formation of new root organs called nodules. Within the nodule, Rhizobiaceae differentiate into nitrogen-fixing bacteroids. However, this symbiotic interaction is time-limited as a result of the initiation of a senescence process, leading to a complete degradation of bacteroids and host plant cells. The increase in proteolytic activity is one of the key features of this process. In this study, we analysed the involvement of two different classes of cysteine proteinases, MtCP6 and MtVPE, in the senescence process of Medicago truncatula nodules. Spatiotemporal expression of MtCP6 and MtVPE was investigated using promoter- β-glucuronidase fusions. Corresponding gene inductions were observed during both developmental and stress-induced nodule senescence. Both MtCP6 and MtVPE proteolytic activities were increased during stress-induced senescence. Down-regulation of both proteinases mediated by RNAi in the senescence zone delayed nodule senescence and increased nitrogen fixation, while their early expression promoted nodule senescence. Using green fluorescent protein fusions, in vivo confocal imaging showed that both proteinases accumulated in the vacuole of uninfected cells or the symbiosomes of infected cells. These data enlighten the crucial role of MtCP6 and MtVPE in the onset of nodule senescence.

An RNA Sequencing Transcriptome Analysis Reveals Novel Insights into Molecular Aspects of the Nitrate Impact on the Nodule Activity of Medicago truncatula

The mechanism through which nitrate reduces the activity of legume nodules is controversial. The objective of the study was to follow Medicago truncatula nodule activity after nitrate provision continuously and to identify molecular mechanisms, which down-regulate the activity of the nodules. Nodule H2 evolution started to decline after about 4 h of nitrate application. At that point in time, a strong shift in nodule gene expression (RNA sequencing) had occurred (1,120 differentially expressed genes). The most pronounced effect was the down-regulation of 127 genes for nodule-specific cysteine-rich peptides. Various other nodulins were also strongly down-regulated, in particular all the genes for leghemoglobins. In addition, shifts in the expression of genes involved in cellular iron allocation and mitochondrial ATP synthesis were observed. Furthermore, the expression of numerous genes for the formation of proteins and glycoproteins with no obvious function in nodules (e.g. germins, patatin, and thaumatin) was strongly increased. This occurred in conjunction with an up-regulation of genes for proteinase inhibitors, in particular those containing the Kunitz domain. The additionally formed proteins might possibly be involved in reducing nodule oxygen permeability. Between 4 and 28 h of nitrate exposure, a further reduction in nodule activity occurred, and the number of differentially expressed genes almost tripled. In particular, there was a differential expression of genes connected with emerging senescence. It is concluded that nitrate exerts rapid and manifold effects on nitrogenase activity. A certain degree of nitrate tolerance might be achieved when the down-regulatory effect on late nodulins can be alleviated.

A correlative study of hydrogen peroxide accumulation after mercury or copper treatment observed in root nodules of Medicago truncatula under light, confocal and electron microscopy

Heavy metal stress affects both, nodulation and nitrogen fixation of legumes. Mercury triggers disturbances in cellular structure and metabolism but its influence on ROS generation is poorly understood. Copper is redox active metal which in opposition to mercury is an essential micronutrient for plants. Excess of copper is cytotoxic, as it participates in ROS generation via Fenton-type reaction.The present work describes changes in hydrogen peroxide (H2O2) accumulation in response to monthly stress caused by mercury (6mg/L HgCl2) or copper (60mg/L CuCl2) in root nodules.H2O2 accumulation viewed with a light microscopy was detected by the use of diaminobenzidine (DAB). 2′,7′-Dichlorofluorescein diacetate (H2DCF-DA) was used as a probe for the intracellular localization of H2O2 with a confocal laser scanning system. H2O2 detection under transmission electron microscopy was performed by the use of cerium method.Histochemical localization and light and confocal microscopy investigations revealed that under Hg or Cu treatments distinct amount of H2O2 accumulated mainly in the interzone and nitrogen-fixing zone. Under normal conditions H2O2 accumulated predominantly in the interzone. Electron microscopy observations showed H2O2 accumulation under Hg or Cu- treatments around peribacteroid membranes of mature symbiosomes located within nitrogen-fixing zone. It should be underlined that under normal conditions H2O2 was not detected at the peribacteroid membranes.The main result of our observations is increased accumulation of H2O2 in response to mercury and copper treatments at the peribacteroidal membranes, to our knowledge shown for the first time. Therefore, our results revealed that an overproduction of H2O2 in response to copper or mercury-treatment may account for lowering of nitrogen fixation rates in heavy-metal affected root nodules.

Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

Growth and nodulation of symbiotic Medicago truncatula at different levels of phosphorus availability.

Medicago truncatula is an important model plant for characterization of P deficiency on leguminous plants at the physiological and molecular levels. Growth optimization of this plant with regard to P supply is the first essential step for elucidation of the role of P in regulation of nodulation. Hence, a study was carried out to address the growth pattern of M. truncatula hydroponically grown at different gradual increases in P levels. The findings revealed that M. truncatula had a narrow P regime, with an optimum P level (12 μM P) which is relatively close to the concentration that induces P toxicity. The accumulated P concentration (2.7 mg g–1 dry matter), which is normal for other crops and legumes, adversely affected the growth of M. truncatula plants. Under P deficiency, M. truncatula showed a higher symbiotic efficiency with Sinorhizobium meliloti 2011 in comparison with S. meliloti 102F51, partially as a result of higher electron allocation to N2 versus H+. The total composition of free amino acids in the phloem was significantly affected by P deprivation. This pattern was found to be almost exclusively the result of the increase in the asparagine level, suggesting that asparagine might be the shoot-derived signal that translocates to the nodules and exerts the down-regulation of nitrogenase activity. Additionally, P deprivation was found to have a strong influence on the contents of the nodule carbon metabolites. While levels of sucrose and succinate tended to decrease, a higher accumulation of malate was observed. These findings have provided evidence that N2 fixation of M. truncatula is mediated through an N feedback mechanism which is closely related to nodule carbon metabolism.

Regulatory patterns of a large family of defensin-like genes expressed in nodules of Medicago truncatula.

Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the largest gene families expressed in the nodules of the model legume Medicago truncatula is the nodule cysteine-rich (NCR) group of defensin-like (DEFL) genes. We used a custom Affymetrix microarray to catalog the expression changes of 566 NCRs at different stages of nodule development. Additionally, bacterial mutants were used to understand the importance of the rhizobial partners in induction of NCRs. Expression of early NCRs was detected during the initial infection of rhizobia in nodules and expression continued as nodules became mature. Late NCRs were induced concomitantly with bacteroid development in the nodules. The induction of early and late NCRs was correlated with the number and morphology of rhizobia in the nodule. Conserved 41 to 50 bp motifs identified in the upstream 1,000 bp promoter regions of NCRs were required for promoter activity. These cis-element motifs were found to be unique to the NCR family among all annotated genes in the M. truncatula genome, although they contain sub-regions with clear similarity to known regulatory motifs involved in nodule-specific expression and temporal gene regulation.

Iron distribution through the developmental stages of Medicago truncatula nodules

Paramount to symbiotic nitrogen fixation (SNF) is the synthesis of a number of metalloenzymes that use iron as a critical component of their catalytical core. Since this process is carried out by endosymbiotic rhizobia living in legume root nodules, the mechanisms involved in iron delivery to the rhizobia-containing cells are critical for SNF. In order to gain insight into iron transport to the nodule, we have used synchrotron-based X-ray fluorescence to determine the spatio-temporal distribution of this metal in nodules of the legume Medicago truncatula with hitherto unattained sensitivity and resolution. The data support a model in which iron is released from the vasculature into the apoplast of the infection/differentiation zone of the nodule (zone II). The infected cell subsequently takes up this apoplastic iron and delivers it to the symbiosome and the secretory system to synthesize ferroproteins. Upon senescence, iron is relocated to the vasculature to be reused by the shoot. These observations highlight the important role of yet to be discovered metal transporters in iron compartmentalization in the nodule and in the recovery of an essential and scarce nutrient for flowering and seed production.