The establishment of an osseointegration is crucial for the long-term stability and functionality of implant materials, and early angiogenesis is the key to successful osseointegration. However, the bioinertness of titanium implants affects osseointegration, limiting their clinical application. In this study, inspired by the rapid polarization of macrophages following the phagocytosis of bacteria, we developed bacteroid cerium oxide particles; these particles were composed of CeO2 and had a size similar to that of Bacillus (0.5 μ m). These particles were constructed on the implant surfaces using a hydrothermal method. In vitro experiments demonstrated that the particles effectively decreased the reactive oxygen species (ROS) levels in macrophages (RAW264.7). Furthermore, these particles exerted effects on M1 macrophage polarization, enhanced nitric oxide (NO) secretion to promote vascular regeneration, and facilitated rapid macrophage transition to the M2 phenotype. Subsequently, the particles facilitated human umbilical vein endothelial cell (HUVEC) migration. In vivo studies showed that these particles rapidly stimulated innate immune responses in animal models, leading to enhanced angiogenesis around the implant and improved osseointegration. In summary, the presence of bacteroid cerium oxide particles on the implant surface regulated and accelerated macrophage polarization, thereby enhancing angiogenesis during the immune response and improving peri-implant osseointegration.

Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress.

Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

Rhizobium leguminosarum bv. viciae (Rlv) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a diaminobutyrate-2-oxoglutarate aminotransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor β-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere.

Triazole fungicides are widely used in agricultural production for plant protection, including pea (Pisum sativum L.). The use of fungicides can negatively affect the legume-Rhizobium symbiosis. In this study, the effects of triazole fungicides Vintage and Titul Duo on nodule formation and, in particular, on nodule morphology, were studied. Both fungicides at the highest concentration decreased the nodule number and dry weight of the roots 20 days after inoculation. Transmission electron microscopy revealed the following ultrastructural changes in nodules: modifications in the cell walls (their clearing and thinning), thickening of the infection thread walls with the formation of outgrowths, accumulation of poly-β-hydroxybutyrates in bacteroids, expansion of the peribacteroid space, and fusion of symbiosomes. Fungicides Vintage and Titul Duo negatively affect the composition of cell walls, leading to a decrease in the activity of synthesis of cellulose microfibrils and an increase in the number of matrix polysaccharides of cell walls. The results obtained coincide well with the data of transcriptomic analysis, which revealed an increase in the expression levels of genes that control cell wall modification and defense reactions. The data obtained indicate the need for further research on the effects of pesticides on the legume-Rhizobium symbiosis in order to optimize their use.

Plant cell differentiation is based on rearrangements of the tubulin cytoskeleton; this is also true for symbiotic nodules. Nevertheless, although for indeterminate nodules (with a long-lasting meristem) the organization of microtubules during nodule development has been studied for various species, for determinate ones (with limited meristem activity) such studies are rare. Here, we investigated bacteroid morphology and dynamics of the tubulin cytoskeleton in determinate nodules of four legume species: Glycine max, Glycine soja, Phaseolus vulgaris, and Lotus japonicus. The most pronounced differentiation of bacteroids was observed in G. soja nodules. In meristematic cells in incipient nodules of all analyzed species, the organization of both cortical and endoplasmic microtubules was similar to that described for meristematic cells of indeterminate nodules. In young infected cells in developing nodules of all four species, cortical microtubules formed irregular patterns (microtubules were criss-crossed) and endoplasmic ones were associated with infection threads and infection droplets. Surprisingly, in uninfected cells the patterns of cortical microtubules differed in nodules of G. max and G. soja on the one hand, and P. vulgaris and L. japonicus on the other. The first two species exhibited irregular patterns, while the remaining two exhibited regular ones (microtubules were oriented transversely to the longitudinal axis of cell) that are typical for uninfected cells of indeterminate nodules. In contrast to indeterminate nodules, in mature determinate nodules of all four studied species, cortical microtubules formed a regular pattern in infected cells. Thus, our analysis revealed common patterns of tubulin cytoskeleton in the determinate nodules of four legume species, and species-specific differences were associated with the organization of cortical microtubules in uninfected cells. When compared with indeterminate nodules, the most pronounced differences were associated with the organization of cortical microtubules in nitrogen-fixing infected cells. The revealed differences indicated a possible transition during evolution of infected cells from anisotropic growth in determinate nodules to isodiametric growth in indeterminate nodules. It can be assumed that this transition provided an evolutionary advantage to those legume species with indeterminate nodules, enabling them to host symbiosomes in their infected cells more efficiently.

In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, \"locked\" infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, \"locked\" infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.

The tubulin cytoskeleton plays an important role in establishing legume-rhizobial symbiosis at all stages of its development. Previously, tubulin cytoskeleton organization was studied in detail in the indeterminate nodules of two legume species, Pisum sativum and Medicago truncatula. General as well as species-specific patterns were revealed. To further the understanding of the formation of general and species-specific microtubule patterns in indeterminate nodules, the tubulin cytoskeleton organization was studied in three legume species (Vicia sativa, Galega orientalis, and Cicer arietinum). It is shown that these species differ in the shape and size of rhizobial cells (bacteroids). Immunolocalization of microtubules revealed the universality of cortical and endoplasmic microtubule organization in the meristematic cells, infected cells of the infection zone, and uninfected cells in nodules of the three species. However, there are differences in the endoplasmic microtubule organization in nitrogen-fixing cells among the species, as confirmed by quantitative analysis. It appears that the differences are linked to bacteroid morphology (both shape and size).

•  A rapid method for detailed analysis of nodule formation has been developed. •  Inoculated root tissues were stained with SYTO 13, a cell-permeant fluorescent nucleic acid-binding dye, and visualized using confocal laser scanning microscopy (CLSM). Structures with high concentrations of DNA and RNA, such as plant cell nuclei and bacteria, labeled strongly. The autofluorescent properties of cell walls made it possible to use CLSM to visualize both plant and rhizobial structures and generate a three-dimensional reconstruction of the root and invading bacteria. •  This method allowed clear observation of stages and structures important in nodule formation, such as rhizobial attachment to root hairs, hair deformation, infection thread ramification, nodule primordium development and nodule cell invasion. Bacteroid structures were easily assessed without the need for fixation that might alter cellular integrity. Plant nodulation mutants with phenotypic differences in thread growth, cellular invasion and plant defense response were also documented. •  Multiple samples can be assessed using detailed microscopy without the need for extensive preparative work, labor-intensive analysis, or the generation of genetically modified samples.

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants; however, they don\'t carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homolog from a pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular, and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small, ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA sequencing analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, an FtsZ2 homolog was commonly found in Rhizobium, Sinorhizobium, and Mesorhizobium spp. but not in closely related nonsymbiotic species. Bacterial two-hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived nodule-specific cysteine-rich peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1 and FtsZ2 causes the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of members of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.