Mastoparan activates calcium spiking analogous to Nod factor-induced responses in Medicago truncatula root hair cells

The rhizobial-derived signaling molecule Nod factor is essential for the establishment of the Medicago truncatula/Sinorhizobium meliloti symbiosis. Nod factor perception and signal transduction in the plant involve calcium spiking and lead to the induction of nodulation gene expression. It has previously been shown that the heterotrimeric G-protein agonist mastoparan can activate nodulation gene expression in a manner analogous to Nod factor activation of these genes and this requires DOESN'T MAKE INFECTIONS3 (DMI3), a calcium- and calmodulin-dependent protein kinase (CCaMK) that is required for Nod factor signaling. Here we show that mastoparan activates oscillations in cytosolic calcium similar but not identical to Nod factor-induced calcium spiking. Mastoparan-induced calcium changes occur throughout the cell, whereas Nod factor-induced changes are restricted to the region associated with the nucleus. Mastoparan-induced calcium spiking occurs in plants mutated in the receptor-like kinases NOD FACTOR PERCEPTION and DMI2 and in the putative cation channel DMI1, which are all required for Nod factor induction of calcium spiking, indicating either that mastoparan functions downstream of these components or that it uses an alternative mechanism to Nod factor for activation of calcium spiking. However, both mastoparan and Nod factor-induced calcium spiking are inhibited by cyclopiazonic acid and n-butanol, suggesting some common mechanisms underpinning these two calcium agonists. The fact that mastoparan and Nod factor both activate calcium spiking and can induce nodulation gene expression in a DMI3-dependent manner strongly implicates CCaMK in the perception and transduction of the calcium signal.     

Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.     

The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling

The symbiotic infection of the model legume Medicago truncatula by Sinorhizobium meliloti involves marked root hair curling, a stage where entrapment of the microsymbiont occurs in a chamber from which infection thread formation is initiated within the root hair. We have genetically dissected these early symbiotic interactions using both plant and rhizobial mutants and have identified a M. truncatula gene, HCL, which controls root hair curling. S. meliloti Nod factors, which are required for the infection process, induced wild-type epidermal nodulin gene expression and root hair deformation in hcl mutants, while Nod factor induction of cortical cell division foci was reduced compared to wild-type plants. Studies of the position of nuclei and of the microtubule cytoskeleton network of hcl mutants revealed that root hair, as well as cortical cells, were activated in response to S. meliloti. However, the asymmetric microtubule network that is typical of curled root hairs, did not form in the mutants, and activated cortical cells did not become polarised and did not exhibit the microtubular cytoplasmic bridges characteristic of the pre-infection threads induced by rhizobia in M. truncatula. These data suggest that hcl mutations alter the formation of signalling centres that normally provide positional information for the reorganisation of the microtubular cytoskeleton in epidermal and cortical cells.     

Proteomic analysis of Medicago truncatula root plastids

Despite the recognized importance of non‐photosynthetic plastids in a wide array of plant processes, the root plastid proteome of soil‐grown plants still remains to be explored. In this study, we used a protocol allowing the isolation of Medicago truncatula root plastids with sufficient protein recovery and purity for their subsequent in‐depth analysis by nanoscale capillary LC‐MS/MS. Besides providing the first picture of a root plastid proteome, the results obtained highlighted the identification of 266 protein candidates whose functional distribution mainly resembled that of wheat endosperm amyloplasts and tobacco proplastids together with displaying major differences to those reported for chloroplasts. Most of the identified proteins have a role in nucleic acid‐related processes (16%), carbohydrate (15%) and nitrogen/sulphur (12%) metabolisms together with stress response mechanisms (10%). It is noteworthy that BLAST searches performed against the proteins reported in different plastidomes allowed detecting 30 putative root plastid proteins for which homologues were previously unsuspected as plastid‐located, most of them displaying a common putative role in participating in the plant cell responses against abiotic and/or biotic stresses. Taken together, the data obtained provide new insights into the functioning of root plastids and reinforce the emerging idea for an important role of these organelles in sustaining plant defence reactions.     

Microtubule dynamics in root hairs of Medicago truncatula

The microtubular cytoskeleton plays an important role in the development of tip-growing plant cells, but knowledge about its dynamics is incomplete. In this study, root hairs of the legume Medicago truncatula have been chosen for a detailed analysis of microtubular cytoskeleton dynamics using GFP-MBD and EB1-YFP as markers and 4D imaging. The microtubular cytoskeleton appears mainly to be composed of bundles which form tracks along which new microtubules polymerise. Polymerisation rates of microtubules are highest in the tip of growing root hairs. Treatment of root hairs with Nod factor and latrunculin B result in a twofold decrease in polymerisation rate. Nonetheless, no direct, physical interaction between the actin filament cytoskeleton and microtubules could be observed. A new picture of how the plant cytoskeleton is organised in apically growing root hairs emerges from these observations, revealing similarities with the organisation in other, non-plant, tip-growing cells.     

Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway

Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.     

LysM domains of Medicago truncatula NFP protein involved in Nod factor perception. Glycosylation state, molecular modeling and docking of chitooligosaccharides and Nod factors

The establishment of the symbiosis between legume plants and rhizobial bacteria depends on the production of rhizobial lipo-chitooligosaccharidic signals (the Nod factors) that are specifically recognized by roots of the host plant. In Medicago truncatula, specific recognition of Sinorhizobium meliloti and its Nod factors requires the NFP (Nod factor perception) gene, which encodes a putative serine/threonine receptor-like kinase (RLK). The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin binding in various bacterial or eukaryotic proteins, respectively. We report here the homology modeling of the three LysM domains of M. truncatula NFP based on the structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosidase D (MltD). Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. Surface analysis and docking calculations performed on the models of the three domains were used to predict the most favored binding modes for chitooligosaccharides and Nod factors. A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. N-glycosylation is not expected to interfere with Nod factor binding in this orientation.     

Root responses of Medicago truncatula plants grown in two different iron deficiency conditions: changes in root protein profile and riboflavin biosynthesis

Iron deficiency is a yield-limiting factor with major implications for field crop production in one-third of the world's agricultural areas, especially those with high soil CaCO(3). In the present work, a two-dimensional gel electrophoresis proteomic approach was combined with a study on the riboflavin synthesis pathway, including qPCR and riboflavin determination, to investigate Fe-deficiency responses in Medicago truncatula plants grown with and without CaCO(3). Iron deficiency caused a de novo accumulation of DMRLs and GTPcII, proteins involved in riboflavin biosynthesis, as well as marked increases in root riboflavin concentrations and in the expression of four genes from the riboflavin biosynthetic pathway. Two novel changes found were the increased accumulation of proteins related to N recycling and protein catabolism. Other identified changes were consistent with previously found increases in glycolysis, TCA cycle, and stress-related processes. All effects were more marked in the presence of CaCO(3). Our results show that the riboflavin biosynthesis pathway was up-regulated at the genomic, proteomic, and metabolomic levels under both Fe-deficiency treatments, especially in the presence of CaCO(3). Results also indicate that N recycling occurs in M. truncatula upon Fe deficiency, possibly constituting an additional anaplerotic N and C source for the synthesis of secondary metabolites, carboxylates, and others.     

microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5′ RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.