An SHR-SCR module specifies legume cortical cell fate to enable nodulation.

Legumes, unlike other plants, have the ability to establish symbiosis with nitrogen-fixing rhizobia. It has been theorized that a unique property of legume root cortical cells enabled the initial establishment of rhizobial symbiosis1-3. Here we show that a SHORTROOT-SCARECROW (SHR-SCR) stem cell program in cortical cells of the legume Medicago truncatula specifies their distinct fate. Regulatory elements drive the cortical expression of SCR, and stele-expressed SHR protein accumulates in cortical cells of M. truncatula but not Arabidopsis thaliana. The cortical SHR-SCR network is conserved across legume species, responds to rhizobial signals, and initiates legume-specific cortical cell division for de novo nodule organogenesis and accommodation of rhizobia. Ectopic activation of SHR and SCR in legumes is sufficient to induce root cortical cell division. Our work suggests that acquisition of the cortical SHR-SCR module enabled cell division coupled to rhizobial infection in legumes. We propose that this event was central to the evolution of rhizobial endosymbiosis.

Medicago truncatula root developmental changes by growth-promoting microbes isolated from Fabaceae, growing on organic farms, involve cell cycle changes and WOX5 gene expression.

MAIN CONCLUSION: Both root nodules and the rhizosphere of Fabaceae plants grown on organic farms are a rich source of bacteria, mainly from the families Enterobacteriaceae and Pseudomonadaceae. The enhanced root system growth in M. truncatula after inoculation with selected bacteria includes an increase of nuclei in the cell cycle S phase and a reduction in phase G2 as well as an enhanced expression of the WOX5 gene. Synthetic fertilizers and pesticides are commonly used to improve plant quality and health. However, it is necessary to look for other efficient and also environmentally safe methods. One such method involves the use of bacteria known as plant growth-promoting bacteria (PGPB). Seventy-two bacterial isolates from the rhizospheric soil and root nodule samples of legumes, including bean, alfalfa, lupine and barrel medic, grown on an organic farm in Western Pomerania (Poland) were screened for their growth-promoting capacities and 38 selected isolates were identified based on 16S rRNA gene sequencing. The analysis showed the isolates to represent 17 strains assigned to 6 families: Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, Rhizobiaceae, Bacillaceae and Alcaligenaceae. Pot experiments showed that 13 strains, capable of producing indole compounds from tryptophan in vitro, could significantly enhance the root and shoot weight of 10-week-old Medicago truncatula seedlings. Compared to non-inoculated seedlings, the root system of inoculated ones was more branched; in addition, the root length, surface area and, especially, the root volume were higher. The 24-h root inoculation with the three selected strains increased the nuclei population in the G1 and S phases, decreased it in the G2 phase and enhanced the WUSCHEL-related Homeobox5 (WOX5) gene expression in root tips and lateral zones. The "arrest" of nuclei in the S phase and the enhancement of the WOX5 gene expression were observed to gradually disappear once the bacterial suspension was rinsed off the seedling roots and the roots were transferred to water for further growth. This study shows that the nodules and rhizosphere of legumes grown on organic farms are a rich source of different PGPB species and provides new data on the ability of these bacteria to interfere with cell cycle and gene expression during the root development.

Transforming compound leaf patterning by manipulating REVOLUTA in Medicago truncatula.

Leaves are derived from the shoot apical meristem with three distinct axes: dorsoventral, proximodistal and mediolateral. Different regulators are involved in the establishment of leaf polarity. Members of the class III homeodomain-leucine zipper (HD-ZIPIII) gene family are critical players in the determination of leaf adaxial identity mediated by microRNA165/166. However, their roles in compound leaf development are still unclear. By screening of a retrotransposon-tagged mutant population of the model legume plant Medicago truncatula, a mutant line with altered leaflet numbers was isolated and characterized. Mutant leaves partially lost their adaxial identity. Leaflet numbers in the mutant were increased along the proximodistal axis, showing pinnate pentafoliate leaves in most cases, in contrast to the trifoliate leaves of the wild type. Detailed characterization revealed that a lesion in a HD-ZIPIII gene, REVOLUTA (MtREV1), resulted in the defects of the mutant. Overexpression of MtMIR166-insensitive MtREV1 led to adaxialized leaves and ectopic leaflets along the dorsoventral axis. Accompanying the abnormal leaf patterning, the free auxin content was affected. Our results demonstrate that MtREV1 plays a key role in determination of leaf adaxial-abaxial polarity and compound leaf patterning, which is associated with proper auxin homeostasis.

Gibberellins negatively regulate the development of Medicago truncatula root system.

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.

Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula.

Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.

Medicago truncatula copper transporter 1 (MtCOPT1) delivers copper for symbiotic nitrogen fixation.

Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper (Cu) transporter would introduce it into the cell to synthesize cupro-proteins. COPT family members in the model legume Medicago truncatula were identified and their expression determined. Yeast complementation assays, confocal microscopy and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the nodule-specific M. truncatula COPT family member. Medicago truncatula genome encodes eight COPT transporters. MtCOPT1 (Medtr4g019870) is the only nodule-specific COPT gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a Cu-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of MtCOPT1 results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a Cu-dependent function, such as cytochrome oxidase activity in copt1-1 bacteroids. These data are consistent with a model in which MtCOPT1 transports Cu from the apoplast into nodule cells to provide Cu for essential metabolic processes associated with symbiotic nitrogen fixation.

Cadmium-induced changes in antioxidative systems and differentiation in roots of contrasted Medicago truncatula lines.

Defense pathways and stress responses induced under Cd stress were illustrated in roots of hydroponically grown Medicago truncatula seedlings. Actually, the ascorbate-glutathione and antioxidative system, secondary metabolism events including peroxidases, phenolic compounds, and lignification launching, and developmental modifications were described. Cd (100 µM) initially increased reactive oxygen species, enhanced antioxidative (total SOD, CAT, and PRX) and ascorbate-glutathione-related metabolism enzymes (APX and MDAR), except in A17 and TN1.11. In agreement with peroxidase enhancement, physiological measurement and in situ observation illustrated soluble phenolic compound accumulation under Cd treatment. However, lignification was restricted to recently created protoxylem elements established in the root tip area, usually constituting the elongation zone. Cell death was increased. In the absence of necrotic reactions, developmental changes including lignin deposition, increase in cellulose and pectin contents, intercellular meatus, and condensed and deformed hairs were noticed in Cd-treated roots.

A symbiotic SNARE protein generated by alternative termination of transcription.

Many microbes interact with their hosts across a membrane interface, which is often distinct from existing membranes. Understanding how this interface acquires its identity has significant implications. In the symbiosis between legumes and rhizobia, the symbiosome encases the intracellular bacteria and receives host secretory proteins important for bacterial development. We show that the Medicago truncatula SYNTAXIN 132 (SYP132) gene undergoes alternative cleavage and polyadenylation during transcription, giving rise to two target-membrane soluble NSF attachment protein receptor (t-SNARE) isoforms. One of these isoforms, SYP132A, is induced during the symbiosis, is able to localize to the peribacteroid membrane, and is required for the maturation of symbiosomes into functional forms. The second isoform, SYP132C, has important functions unrelated to symbiosis. The SYP132A sequence is broadly found in flowering plants that form arbuscular mycorrhizal symbiosis, an ancestral mutualism between soil fungi and most land plants. SYP132A silencing severely inhibited arbuscule colonization, indicating that SYP132A is an ancient factor specifying plant-microbe interfaces.

Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signaling pathway and ETHYLENE INSENSITIVE2 (EIN2)/SICKLE (SKL), but acts independently of SUPER NUMERIC NODULES. MtCEP1-dependent inhibition of lateral root development acts through an EIN2-independent mechanism. MtCEP1 increases nodulation by promoting rhizobial infections, the developmental competency of roots for nodulation, the formation of fused nodules, and an increase in frequency of nodule development that initiates at proto-phloem poles. These phenotypes are similar to those of the ein2/skl mutant and support that MtCEP1 modulates EIN2-dependent symbiotic responses. Accordingly, MtCEP1 counteracts the reduction in nodulation induced by increasing ethylene precursor concentrations, and an ethylene synthesis inhibitor treatment antagonizes MtCEP1 root phenotypes. MtCEP1 also inhibits the development of EIN2-dependent pseudonodule formation. Finally, mutants affecting the COMPACT ROOT ARCHITECTURE2 (CRA2) receptor, which is closely related to the Arabidopsis CEP Receptor1, are unresponsive to MtCEP1 effects on lateral root and nodule formation, suggesting that CRA2 is a CEP peptide receptor mediating both organogenesis programs. In addition, an ethylene inhibitor treatment counteracts the cra2 nodulation phenotype. These results indicate that MtCEP1 and its likely receptor, CRA2, mediate nodulation and lateral root development through different pathways.

Fluorescence Imaging of the Cytoskeleton in Plant Roots.

During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.

Cell length instead of cell number becomes the predominant factor contributing to hypocotyl length genotypic differences under abiotic stress in Medicago truncatula.

Hypocotyl elongation in the dark is a crucial process to ensure seedling emergence. It relies both on the cell number and cell length. The contribution of these two factors to the maximal hypocotyl length and the impact of environmental conditions on this contribution are not known. This is surprising considering the agronomic and economical importance of seedling emergence in crop establishment. Using 14 genotypes from a nested core collection representing Medicago truncatula (barrel medic) natural variation, we investigated how epidermal cell number and cell length contribute to hypocotyl length under optimal, low temperature (8°C) and water deficit (-0.50 MPa) conditions. Both cell number and length vary according to genotypes and contribute to maximal hypocotyl length differences between genotypes. This contribution, however, depends on growth conditions. Cell number is the major contributor under optimal conditions (60%) whereas cell length becomes the major determinant under stress. Maximal hypocotyl length is correlated with hypocotyl elongation rate under both stresses but not under optimal condition, revealing contrasted genotypes for cell elongation capacity under stress. To identify the genetic regulators determining cell number and cell length, quantitative trait loci (QTLs) were detected using a recombinant inbred lines population exhibiting segregation in maximal hypocotyl length. Two QTLs controlling cell number and three QTLs controlling cell length at low temperature were detected. One QTL for cell number and two for cell length were found to be associated with hypocotyl length under low temperature. This study provides new information to improve seedling emergence under abiotic stress.

The Medicago truncatula MtRbohE gene is activated in arbusculated cells and is involved in root cortex colonization.

MAIN CONCLUSION: Our study demonstrated that the NAPDH oxidase gene MtRbohE is expressed in arbusculated cells and plays a role in arbuscule development. Plant NADPH oxidases, known as respiratory burst oxidase homologs (RBOH), belong to a multigenic family that plays an important role in the regulation of plant development and responses to biotic and abiotic stresses. In this study, we monitored the expression profiles of five Rboh genes (MtRbohA, MtRbohB, MtRbohE, MtRbohG, MtRbohF) in the roots of the model species Medicago truncatula upon colonization by arbuscular mycorrhizal fungi. A complementary cellular and molecular approach was used to monitor changes in mRNA abundance and localize transcripts in different cell types from mycorrhizal roots. Rboh transcript levels did not drastically change in total RNA extractions from whole mycorrhizal and non-mycorrhizal roots. Nevertheless, the analysis of laser microdissected cells and Agrobacterium rhizogenes-transformed roots expressing a GUS transcriptional fusion construct highlighted the MtRbohE expression in arbuscule-containing cells. Furthermore, the down regulation of MtRbohE by an RNAi approach generated an altered colonization pattern in the root cortex, when compared to control roots, with fewer arbuscules and multiple penetration attempts. Altogether our data indicate a transient up-regulation of MtRbohE expression in cortical cells colonized by arbuscules and suggest a role for MtRbohE in arbuscule accommodation within cortical cells.

The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root.

Biological nitrogen fixation in legumes occurs in nodules that are initiated in the root cortex following Nod factor recognition at the root surface, and this requires coordination of diverse developmental programs in these different tissues. We show that while early Nod factor signaling associated with calcium oscillations is limited to the root surface, the resultant activation of Nodule Inception (NIN) in the root epidermis is sufficient to promote cytokinin signaling and nodule organogenesis in the inner root cortex. NIN or a product of its action must be associated with the transmission of a signal between the root surface and the cortical cells where nodule organogenesis is initiated. NIN appears to have distinct functions in the root epidermis and the root cortex. In the epidermis, NIN restricts the extent of Early Nodulin 11 (ENOD11) expression and does so through competitive inhibition of ERF Required for Nodulation (ERN1). In contrast, NIN is sufficient to promote the expression of the cytokinin receptor Cytokinin Response 1 (CRE1), which is restricted to the root cortex. Our work in Medicago truncatula highlights the complexity of NIN action and places NIN as a central player in the coordination of the symbiotic developmental programs occurring in differing tissues of the root that combined are necessary for a nitrogen-fixing symbiosis.

Symbiosis dependent accumulation of primary metabolites in arbuscule-containing cells.

BACKGROUND: The arbuscular mycorrhizal symbiosis is characterized by the presence of different symbiotic structures and stages within a root system. Therefore tools allowing the analysis of molecular changes at a cellular level are required to reveal insight into arbuscular mycorrhizal (AM) symbiosis development and functioning. RESULTS: Here we describe the analysis of metabolite pools in arbuscule-containing cells, which are the site of nutrient transfer between AM fungus and host plant. Laser capture microdissection (LCM) combined with gas chromatography mass spectrometry (GC-EI/TOF-MS) enabled the analysis of primary metabolite levels,which might be of plant or fungal origin, within these cells. CONCLUSIONS: High levels of the amino acids, aspartate, asparagine, glutamate, and glutamine, were observed in arbuscule-containing cells. Elevated amounts of sucrose and the steady-state of hexose levels indicated a direct assimilation of monosaccharides by the fungal partner.

The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula.

Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula.

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Remodeling of the infection chamber before infection thread formation reveals a two-step mechanism for rhizobial entry into the host legume root hair.

In many legumes, root entry of symbiotic nitrogen-fixing rhizobia occurs via host-constructed tubular tip-growing structures known as infection threads (ITs). Here, we have used a confocal microscopy live-tissue imaging approach to investigate early stages of IT formation in Medicago truncatula root hairs (RHs) expressing fluorescent protein fusion reporters. This has revealed that ITs only initiate 10 to 20 h after the completion of RH curling, by which time major modifications have occurred within the so-called infection chamber, the site of bacterial entrapment. These include the accumulation of exocytosis (M. truncatula Vesicle-Associated Membrane Protein721e)- and cell wall (M. truncatula EARLY NODULIN11)-associated markers, concomitant with radial expansion of the chamber. Significantly, the infection-defective M. truncatula nodule inception-1 mutant is unable to create a functional infection chamber. This underlines the importance of the NIN-dependent phase of host cell wall remodeling that accompanies bacterial proliferation and precedes IT formation, and leads us to propose a two-step model for rhizobial infection initiation in legume RHs.

Genome-wide association of drought-related and biomass traits with HapMap SNPs in Medicago truncatula.

Improving drought tolerance of crop plants is a major goal of plant breeders. In this study, we characterized biomass and drought-related traits of 220 Medicago truncatula HapMap accessions. Characterized traits included shoot biomass, maximum leaf size, specific leaf weight, stomatal density, trichome density and shoot carbon-13 isotope discrimination (δ(13) C) of well-watered M. truncatula plants, and leaf performance in vitro under dehydration stress. Genome-wide association analyses were carried out using the general linear model (GLM), the standard mixed linear model (MLM) and compressed MLM (CMLM) in TASSEL, which revealed significant overestimation of P-values by CMLM. For each trait, candidate genes and chromosome regions containing SNP markers were found that are in significant association with the trait. For plant biomass, a 0.5 Mbp region on chromosome 2 harbouring a plasma membrane intrinsic protein, PIP2, was discovered that could potentially be targeted to increase dry matter yield. A protein disulfide isomerase-like protein was found to be tightly associated with both shoot biomass and leaf size. A glutamate-cysteine ligase and an aldehyde dehydrogenase family protein with Arabidopsis homologs strongly expressed in the guard cells were two of the top genes identified by stomata density genome-wide association studies analysis.

Multiple steps control immunity during the intracellular accommodation of rhizobia.

Medicago truncatula belongs to the legume family and forms symbiotic associations with nitrogen fixing bacteria, the rhizobia. During these interactions, the plants develop root nodules in which bacteria invade the plant cells and fix nitrogen for the benefit of the plant. Despite massive infection, legume nodules do not develop visible defence reactions, suggesting a special immune status of these organs. Some factors influencing rhizobium maintenance within the plant cells have been previously identified, such as the M. truncatula NCR peptides whose toxic effects are reduced by the bacterial protein BacA. In addition, DNF2, SymCRK, and RSD are M. truncatula genes required to avoid rhizobial death within the symbiotic cells. DNF2 and SymCRK are essential to prevent defence-like reactions in nodules after bacteria internalization into the symbiotic cells. Herein, we used a combination of genetics, histology and molecular biology approaches to investigate the relationship between the factors preventing bacterial death in the nodule cells. We show that the RSD gene is also required to repress plant defences in nodules. Upon inoculation with the bacA mutant, defence responses are observed only in the dnf2 mutant and not in the symCRK and rsd mutants. In addition, our data suggest that lack of nitrogen fixation by the bacterial partner triggers bacterial death in nodule cells after bacteroid differentiation. Together our data indicate that, after internalization, at least four independent mechanisms prevent bacterial death in the plant cell. These mechanisms involve successively: DNF2, BacA, SymCRK/RSD and bacterial ability to fix nitrogen.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process.