Comprehensive analysis of phenotype, microstructure and global transcriptional profiling to unravel the effect of excess copper on the symbiosis between nitrogen-fixing bacteria and Medicago lupulina.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. However, knowledge of the molecular mechanisms, especially the microstructure and global transcriptional profiling, of the symbiosis process under heavy metal contamination is limited. In this study, a heavy metal-tolerant legume, Medicago lupulina, was treated with different concentrations of copper (Cu). The results showed that the early infection process was inhibited and the nodule ultrastructure was changed under 200 mg kg-1 Cu stress. Most infection threads (ITs) were prevented from entering the nodule cells, and few rhizobia were released into the host cells, in which thickening of the plant cell wall and IT wall was observed, demonstrating that rhizobial invasion was inhibited under Cu stress. RNA-seq analysis indicated that a strong shift in gene expression occurred (3257 differentially expressed genes, DEGs). The most pronounced effect was the upregulation of a set of 71 of 73 DEGs for nodule-specific cysteine-rich peptides, which have been shown to control the terminal differentiation of rhizobia in the nodules and to have antimicrobial activity. Various genes for metal transport, chelation binding and antioxidant defence were regulated. In particular, the DEGs for Cu trafficking and detoxification were induced during nodule formation. The DEGs for ethylene (ET) biosynthesis and signalling were also differentially expressed during nodulation, suggesting that the inhibition of nodulation by Cu occurred partially through ET signalling. Furthermore, the genes related to the cell wall were mostly upregulated and most likely involved in cell wall thickening. These findings provide an integrated understanding of the effects of Cu on legume nodule symbiosis at the molecular and phenotypic levels.

[Development of Arbuscular Mycorrhiza in Highly Responsive and Mycotrophic Host Plant-Black Medick (Medicago lupulina L.)].

The main phases of arbuscular mycorrhiza (AM) development were analyzed in black medick (Medicago lupulina) with Glomus intraradices. Methods of light and transmission electron microscopy were used to investigate AM. The first mycorrhization was identified on the seventh day after sowing. M. lupulina with AM-fungus Glomus intraradices formed Arum type of AM. Roots of black medick at fruiting stage (on the 88th day) were characterized by the development of forceful mycelium. The thickness of mycelium was comparable with the vascular system of root central cylinder. The development of vesicules into intraradical spores was shown. Micelium, arbuscules, and vesicules developed in close vicinity to the division zone of root tip. This might be evidence of an active symbiotic interaction between partners. All stages of fungal development and breeding, including intraradical spores (in inter-cellular matrix of root cortex), were identified in the roots of black medick, which indicated an active utilization of host plant nutrient substrates by the mycosymbiont. Plant cell cytoplasm extension was identified around young arbuscular branches but not for intracellular hyphae. The presence of active symbiosis was confirmed by increased accumulation of phosphorus in M. lupulina root tissues under conditions of G. intraradices inoculation and low phosphorus level in the soil. Thus, black medick cultivar-population can be characterized as an ecologically obligate mycotrophic plant under conditions of low level of available phosphorus in the soil. Specific features of AM development in intensively mycotrophic black medick, starting from the stage of the first true leaf until host plant fruiting, were evaluated. The obtained plant-microbe system is a perspective model object for further ultracytological and molecular genetic studies of the mechanisms controlling arbuscular mycorrhiza symbiotic efficiency, including selection and investigation of new symbiotic plant mutants.

The reticulate history of Medicago (Fabaceae).

The phylogenetic history of Medicago was examined for 60 accessions from 56 species using two nuclear genes (CNGC5 and beta-cop) and one mitochondrial region (rpS14-cob). The results of several analyses revealed that extensive robustly supported incongruence exists among the nuclear genes, the cause of which we seek to explain. After rejecting several processes, hybridization and lineage sorting of ancestral polymorphisms remained as the most likely factors promoting incongruence. Using coalescence simulations, we rejected lineage sorting alone as an explanation of the differences among gene trees. The results indicate that hybridization has been common and ongoing among lineages since the origin of Medicago. Coalescence provides a good framework to test the causes of incongruence commonly seen among gene trees but requires knowledge of effective population sizes and generation times. We estimated the effective population size at 240,000 individuals and assumed a generation time of 1 year in Medicago (many are annual plants). A sensitivity analysis showed that our conclusions remain unchanged using a larger effective population size and/or longer generation time.

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress.

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.

Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules.

In Medicago nodules, endoreduplication cycles and ploidy-dependent cell enlargement occur during the differentiation of bacteroid-containing nitrogen-fixing symbiotic cells. These events are accompanied by the expression of ccs52A, a plant ortholog of the yeast and animal cdh1/srw1/fzr genes, acting as a substrate-specific activator of the anaphase-promoting complex (APC) ubiquitin ligase. Because CCS52A is involved in the transition of mitotic cycles to endoreduplication cycles, we investigated the importance of somatic endoploidy and the role of the M. truncatula ccs52A gene in symbiotic cell differentiation. Transcription analysis and ccs52A promoter-driven beta-glucuronidase activity in transgenic plants showed that ccs52A was dispensable for the mitotic cycles and nodule primordium formation, whereas it was induced before nodule differentiation. The CCS52A protein was present in the nucleus of endoreduplication-competent cells, indicating that it may activate APC constitutively during the endoreduplication cycles. Downregulation of ccs52A in transgenic M. truncatula plants drastically affected nodule development, resulting in lower ploidy, reduced cell size, inefficient invasion, and the maturation of symbiotic cells, accompanied by early senescence and finally the death of both the bacterium and plant cells. Thus, ccs52A expression is essential for the formation of large highly polyploid symbiotic cells, and endoreduplication is an integral part of normal nodule development.

Calcium oxalate crystal morphology mutants from Medicago truncatula.

Plants accumulate crystals of calcium oxalate in a variety of shapes and sizes. The mechanism(s) through which a plant defines the morphology of its crystals remains unknown. To gain insight into the mechanisms regulating crystal shapes, we conducted a mutant screen to identify the genetic determinants. This is the first reported mutant screen dedicated to the identification of crystal morphology mutants. A single leaf was harvested from individual Medicago truncatula L. plants that had been chemically mutagenized. Each leaf was visually inspected, using crossed-polarized light microscopy, for alterations in crystal shape and size. Seven different crystal morphology defective ( cmd) mutants were identified. Six cmd mutants were recessive and one dominant. Genetic analysis of the six recessive mutants suggested that each mutant was affected at a different locus. Each cmd mutant represents a new locus different than any previously identified. The plant phenotype of the cmd mutants appeared similar to that of the wild type in overall growth and development. This observation, coupled with the finding that several of the mutants had drastically altered the amount of calcium they partition into the oxalate crystal, questions current hypotheses regarding crystal function. Comparisons between the mutant crystals and those present in other legumes indicated the likelihood that simple point mutations contributed to the evolution of the variations in prismatic crystal shapes commonly observed in these plants today. The availability of cmd mutants provides the opportunity to investigate aspects of crystal shape and size that have been recalcitrant to previous approaches.

Microtubule organization in root cells of Medicago truncatula during development of an arbuscular mycorrhizal symbiosis with Glomus versiforme.

The colonization of plants by arbuscular mycorrhizal fungi has been shown to induce changes in cytoplasmic organization and morphology of root cells. Because of their role in a variety of cellular functions in plants, it is likely that microtubules are involved either in the signaling events leading to the establishment of the symbiosis or in changes in host cell morphology and cytoplasmic architecture. Recent studies of the arbuscular mycorrhizal symbiosis have shown that root cortical cells reorganize their microtubules upon colonization. These studies, however, have focused primarily on the cells containing hyphal coils or arbuscules and did not include descriptions of microtubule changes in adjacent cells. To probe further into the potential role of the microtubule cytoskeleton in the establishment of arbuscular mycorrhizal symbiosis, we examined the three-dimensional arrangement of microtubules in roots of the model legume Medicago truncatula colonized by the arbuscular mycorrhizal fungus Glomus versiforme by indirect immunofluorescence and confocal microscopy. Our data show extensive remodeling of the microtubule cytoskeleton from the early stages of arbuscule development until arbuscule collapse and senescence. While confirming some of the microtubule patterns shown in other mycorrhizal systems, our results also reveal that cortical cells adjacent to those containing arbuscules or adjacent to intercellular hyphae reorganize their microtubules. This indicates that the cortical cells initiate the modification of their cytoskeleton prior to entry of the fungus and is consistent with signal exchange between the symbionts prior to fungal penetration of the cells.

Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobium-legume symbiosis.

A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.