Artemii P. Gorshkov

The cellular mechanisms underlying nodulation in peanut (Arachis hypogaea L.), which proceeds via an infection-thread-independent intercellular pathway, remain poorly understood relative to those in model legumes. Because microtubules provide the intracellular framework for organelle positioning, nuclear migration, and cell-wall remodeling, they are likely to play a central role in the host-cell reorganization required for rhizobial accommodation. This study examined the spatial and temporal reorganization of microtubules during nodule development in peanut cv. ‘Valencia 433’ inoculated with Bradyrhizobium ottawaense strain RCAM104, following host-cell remodeling from early primordia to mature nodules. Prior to rhizobial entry, cortical microtubules in primordium cells displayed a regular arrangement (transversely oriented microtubules arranged perpendicularly to the longitudinal axis of the cell), which became locally disorganized at bacterial entry sites, accompanied by the presence of the host nucleus near the penetration site. During host cell mitosis, symbiosomes exhibited a coordinated redistribution that aligned with mitotic spindle and phragmoplast dynamics. In young infected cells, cortical microtubules retained a regular pattern, whereas endoplasmic microtubules formed a randomly oriented network. Upon bacteroid differentiation from the U-type to the S-type morphology, cortical microtubules reorganized into an irregular, randomly oriented network. These findings demonstrate that early microtubule responses to intercellular entry share features with conserved symbiotic penetration mechanisms. In contrast, the subsequent shift from a regular to an irregular cortical array—partially resembling that observed in indeterminate nodules—correlates with bacteroid morphological differentiation, highlighting unique cellular dynamics in peanut nodules.

Symbiotic interface plays a crucial role in symbiosis development and stability. In this study, the progressive remodeling of symbiotic interface components, such as pectins, arabinogalactan proteins, bacterial lipopolysaccharides, and others, were analyzed in pea (Pisum sativum L.) nodules induced with siх strains of Rhizobium laguerreae. Some strains induced effective nodules while others ineffective ones. The organization of the symbiotic interface in ineffective nodules differed from that in effective ones. Indeed, ineffective nodules were characterized by reduced pectin modification activity and lack of de-esterified homogalacturonan accumulation. Additionally, abnormal localization of arabinogalactan proteins, arabinogalactan protein-extensin, and callose was observed in response to certain rhizobia strains, indicating the involvement of these components in defense mechanisms during symbiosis. Transcriptome analysis of ineffective nodules revealed activation of plant defense responses and responses to the biotic stresses. Thus, abnormalities in the organization of the symbiotic interface were revealed, accompanying ineffective interaction of pea with R. laguerreae strains.

The study of wild relatives of crop legumes offers an inexhaustible source of useful properties and microorganisms for agriculture. In this study, we morphologically examined vavilovia (Vavilovia formosa (Steven) Fed.) nodules induced by strains of Rhizobium ruizarguesonis RCAM1026, Rhizobium leguminosarum sv. viciae TOM, as well as R. leguminosarum sv. viciae strains Vaf-12 and Vaf-108, isolated from nodules of wild-type plants of V. formosa. The nodules induced by R. leguminosarum sv. viciae strain Vaf-12 maintained histological and ultrastructural organization typical for indeterminate nodules. Different ultrastructural abnormalities were revealed in nodules induced by the other strains. When using the R. leguminosarum sv. viciae strain Vaf-108, pinkish nodules formed, in which a senescence zone developed in four weeks after inoculation. Furthermore, small brownish pseudonodules were also formed. In the nodules induced by the R. leguminosarum sv. viciae strain TOM bacteroids rapidly degraded and were excluded from the cytoplasm into the vacuole. Nodules induced by the R. ruizarguesonis strain 1026 were characterized with excessive accumulation of starch grains in mature infected cells.

Tubulin cytoskeleton rearrangements play an important role in the cell differentiation of symbiotic nodules in legumes. However, the organization of the tubulin cytoskeleton has been investigated only for four legume species forming determinate nodules (with limited nodule meristem activity). In this study, microtubule organization was studied in three species (Vigna radiata, V. unguiculata, and Lotus corniculatus) with determinate nodules using confocal laser scanning microscopy and quantitative analyses. Histological organization in young nodules of V. radiata and V. unguiculata resembled the recently reported zonation in young nodules of Glycine max. In addition, bacteroids in nodules of these species were significantly enlarged compared to free-living bacteria. Organization of endoplasmic and cortical microtubules in young infected cells and uninfected cells and that of cortical microtubules in nitrogen-fixing cells demonstrated general patterns for determinate nodules, whereas endoplasmic microtubules in nitrogen-fixing cells showed species-specific patterns. Thus, the presence of both general and species-specific patterns of tubulin cytoskeleton organization was confirmed in determinate nodules.

Insecticides are used commonly in agricultural production to defend plants, including legumes, from insect pests. It is a known fact that insecticides can have a harmful effect on the legume–rhizobial symbiosis. In this study, the effects of systemic seed treatment insecticide Imidor Pro (imidacloprid) and foliar insecticide Faskord (alpha-cypermethrin) on the structural organization of pea (Pisum sativum L.) nodules and their transcriptomic activity were investigated. The plants were treated as recommended by the manufacturer (10 mg/mL for Imidor Pro and 50 µg/mL for Faskord) and twofold concentrations were used for both insecticides. Insecticides had no visible effect on the growth of pea plants. The nodules also showed no visible changes, except for the variant treated with twofold concentration of Imidor Pro. However, the dry weight of shoots and roots differed significantly in insecticide-treated plants compared to untreated plants in almost all treatments. The number of nodules decreased in variants with Imidor Pro treatment. At the ultrastructural level, both insecticides caused cell wall deformation, poly-β-hydroxybutyrate accumulation in bacteroids, expansion of the peribacteroid space in symbiosomes, and inclusions in vacuoles. Treatment with Faskord caused chromatin condensation in nucleus. Imidor Pro treatment caused hypertrophy of infection droplets by increasing the amount of matrix, as confirmed by immunofluorescence analysis of extensins. Transcriptome analysis revealed upregulation of expression of a number of extensin-like protein-coding genes in nodules after the Imidor Pro treatment. Overall, both insecticides caused some minor changes in the legume–rhizobial system when used at recommended doses, but Faskord, an enteric contact insecticide, has fewer negative effects on symbiotic nodules and legume plants; of these two insecticides, it is preferred in pea agricultural production.

Hydrogen peroxide (H₂O₂) in plants is produced in relatively large amounts and plays a universal role in plant defense and physiological responses, including the regulation of growth and development. In the Rhizobium–legume symbiosis, hydrogen peroxide plays an important signaling role throughout the development of this interaction. In the functioning nodule, H₂O₂ has been shown to be involved in bacterial differentiation into the symbiotic form and in nodule senescence. In this study, the pattern of H₂O₂ accumulation in pea (Pisum sativum L.) wild-type and mutant nodules blocked at different stages of the infection process was analyzed using a cytochemical reaction with cerium chloride. The observed dynamics of H₂O₂ deposition in the infection thread walls indicated that the distribution of H₂O₂ was apparently related to the stiffness of the infection thread wall. The dynamics of H₂O₂ accumulation was traced, and its patterns in different nodule zones were determined in order to investigate the relationship of H₂O₂ localization and distribution with the stages of symbiotic nodule development in P. sativum. The patterns of H₂O₂ localization in different zones of the indeterminate nodule have been partially confirmed by comparative analysis on mutant genotypes.

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.

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.

Chinese liquorice (Glycyrrhiza uralensis Fisch. ex DC.) is widely used in the food industry and as a medicine. Like other legumes, G. uralensis forms symbiotic nodules. However, the structural organization of G. uralensis nodules is poorly understood. In this study, we analyzed the histological and ultrastructural organization and dynamics of the tubulin cytoskeleton in various cells from different histological zones of indeterminate nodules formed by two strains of Mesorhizobium sp. The unusual walls of infection threads and formation of multiple symbiosomes with several swollen bacteroids were observed. A large amount of poly-β-hydroxybutyrate accumulated in the bacteroids, while the vacuoles of meristematic and uninfected cells contained drop-shaped osmiophilic inclusions. Immunolocalization of the tubulin cytoskeleton and quantitative analysis of cytoskeletal elements revealed patterns of cortical microtubules in meristematic, infected and uninfected cells, and of endoplasmic microtubules associated with infection structures, typical of indeterminate nodules. The intermediate pattern of endoplasmic microtubules in infected cells was correlated with disordered arrangement of symbiosomes. Thus, analysis of the structural organization of G. uralensis nodules revealed some ancestral features more characteristic of determinate nodules, demonstrating the evolutionary closeness of G. uralensis nodulation to more ancient members of the legume family.