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A new method for the synthesis of azido-propargyloxy derivatives of 1,3,5-triazine has been developed utilizing the nitrosation of hydrazyno-1,3,5-triazines. New hydrazines (2-hydrazino-4,6-bis(propargyloxy)-1,3,5-triazine and 2,4-dihydrazino-6-propargyloxy-1,3,5-triazine) were synthesized and characterized via FTIR, NMR spectroscopy and elemental analysis. The hyperbranched polymers with azide (diazide monomer) and propargyloxy terminal groups were obtained via the azide-alkyne polycycloaddition reaction of diazide and monoazide AB2-type monomers. The antibacterial activity against Escherichia coli bacteria of 2,4,6-trispropargyloxy-1,3,5-triazine, 2-azido-4,6-bispropargyloxy-1,3,5-triazine, and 2,4-diazido-6-propargyloxy-1,3,5-triazine and their hyperbranched polymers was studied. Only 2,4-diazido-6-propargyloxy-1,3,5-triazine has weak antibacterial activity in comparison with ampicillin. The cytotoxicity of these compounds against M-HeLa, FetMSC, and Vero cell lines was also studied. 2,4,6-trispropargyloxy-1,3,5-triazine does not show any cytotoxic effect (IC50 ≥ 280 µM). It was shown that the presence of an azide group in the compound directly affects the cytotoxic effect. Hyperbranched polymers have a less cytotoxic effect against M-HeLa (IC50 > 100) in comparison with monomers (IC50 = 90–99 µM). This makes it possible to use these polymers as the basis for biocompatible materials in biomedical applications. [ABSTRACT FROM AUTHOR]

Different components of the symbiotic interface play an important role in providing positional information during rhizobial infection and nodule development: successive changes in cell morphology correspond to subsequent changes in the molecular architecture of the apoplast and the associated surface structures. The localisation and distribution of pectins, xyloglucans, and cell wall proteins in symbiotic nodules of Pisum sativum and Medicago truncatula were studied using immunofluorescence and immunogold analysis in wild-type and ineffective mutant nodules. As a result, the ontogenetic changes in the symbiotic interface in the nodules of both species were described. Some differences in the patterns of distribution of cell wall polysaccharides and proteins between wild-type and mutant nodules can be explained by the activation of defence reaction or premature senescence in mutants. The absence of fucosylated xyloglucan in the cell walls in the P. sativum nodules, as well as its predominant accumulation in the cell walls of uninfected cells in the M. truncatula nodules, and the presence of the rhamnogalacturonan I (unbranched) backbone in meristematic cells in P. sativum can be attributed to the most striking species-specific features of the symbiotic interface. [ABSTRACT FROM AUTHOR]

Pisum sativum symbiotic mutant SGEFix−-2 carries the sym33-3 allele of the gene Sym33, encoding transcription factor PsCYCLOPS/PsIPD3. Previously, strong host cell defence reactions were identified in nodules of this mutant. In the present study, new manifestations of defence reactions were revealed in 28-day-old white nodules in which bacterial release had occurred. These nodules were investigated using histochemical staining of pectin and suberin and by immunogold localisation of three components of pectin: highly methyl-esterified homogalacturonan (HG) recognised by monoclonal antibody JIM7, low methyl-esterified HG recognised by JIM5 and linear (1–4)-β-D-galactan side-chain of rhamnogalacturonan I (RG I) recognised by LM5. In the mutant, but not in the wild-type, cell wall material was deposited around the vacuole in the uninfected cells, in cells containing infection threads and in the infected cells. The deposits around the vacuole were marked with JIM7 and LM5 antibodies but not with JIM5, suggesting that they contain newly formed cell wall material. Deposition was accompanied by suberin accumulation. This is the first report that deposition of cell wall material around the vacuole may be associated with the defence reaction in ineffective nodules. In addition, hypertrophic infection droplets labelled with JIM7 were identified. In the matrix of some infection threads, RG I recognised a pectic gel component. Callose deposits in the cell walls and in the walls of infection threads were occasionally observed. The observations suggest that an important function of transcriptional factor CYCLOPS/IPD3 is the suppression of defence reactions during establishment of the legume-rhizobial symbiosis. [ABSTRACT FROM AUTHOR]

Infection of host cells by nitrogen-fixing soil bacteria, known as rhizobia, involves the progressive remodelling of the plant–microbe interface. This process was examined by using monoclonal antibodies to study the subcellular localisation of pectins and arabinogalactan proteins (AGPs) in wild-type and ineffective nodules of Pisum sativum and Medicago truncatula. The highly methylesterified homogalacturonan (HG), detected by monoclonal antibody JIM7, showed a uniform localisation in the cell wall, regardless of the cell type in nodules of P. sativum and M. truncatula. Low methylesterified HG, recognised by JIM5, was detected mainly in the walls of infection threads in nodules of both species. The galactan side chain of rhamnogalacturonan I (RG-I), recognised by LM5, was present in the nodule meristem in both species and in the infection thread walls in P. sativum, but not in M. truncatula. The membrane-anchored AGP recognised by JIM1 was observed on the plasma membrane in nodules of P. sativum and M. truncatula. In P. sativum, the AGP epitope recognised by JIM1 was present on mature symbiosome membranes of wild-type nodules, but JIM1 labelling was absent from symbiosome membranes in the mutant Sprint-2Fix− (sym31) with undifferentiated bacteroids, suggesting a possible involvement of AGP in the maturation of symbiosomes. Thus, the common and species-specific traits of cell wall remodelling during nodule differentiation were demonstrated. [ABSTRACT FROM AUTHOR]

Plant symbiotic mutants are useful tool to uncover the molecular-genetic mechanisms of nodule senescence. The pea (Pisum sativum L.) mutants SGEFix−-1 (sym40), SGEFix−-3 (sym26), and SGEFix−-7 (sym27) display an early nodule senescence phenotype, whereas the mutant SGEFix−-2 (sym33) does not show premature degradation of symbiotic structures, but its nodules show an enhanced immune response. The nodules of these mutants were compared with each other and with those of the wild-type SGE line using seven marker genes that are known to be activated during nodule senescence. In wild-type SGE nodules, transcript levels of all of the senescence-associated genes were highest at 6 weeks after inoculation (WAI). The senescence-associated genes showed higher transcript abundance in mutant nodules than in wild-type nodules at 2 WAI and attained maximum levels in the mutant nodules at 4 WAI. Immunolocalization analyses showed that the ethylene precursor 1-aminocyclopropane-1-carboxylate accumulated earlier in the mutant nodules than in wild-type nodules. Together, these results showed that nodule senescence was activated in ineffective nodules blocked at different developmental stages in pea lines that harbor mutations in four symbiotic genes. [ABSTRACT FROM AUTHOR]

The nitrogen-fixing nodule is a unique ecological niche for rhizobia, where microaerobic conditions support functioning of the main enzyme of nitrogen fixation, nitrogenase, which is highly sensitive to oxygen. To accommodate bacteria in a symbiotic nodule, the specialised infected cells increase in size owing to endoreduplication and are able to shelter thousands of bacteria. Bacteria are isolated from the cytoplasm of the plant cell by a membrane-bound organelle-like structure termed the symbiosome. It is enclosed by a symbiosome membrane, mainly of plant origin but with some inclusion of bacterial proteins. Within the symbiosome, bacterial cells differentiate into bacteroids a form that is specialised for nitrogen fixation. In this review, we briefly summarise recent advances in studies of differentiation both of symbiosomes and of the infected cells that accommodate them. We will consider the role of CCS52A, DNA topoisomerase VI, tubulin cytoskeleton rearrangements in differentiation of infected cells, the fate of the vacuole, and the distribution of symbiosomes in the infected cells.Wewill also consider differentiation of symbiosomes, paying attention to the role ofNCRpeptides, vesicular transport to symbiosomes, and mutant analysis of symbiosome development in model and crop legumes. Finally, we conclude that mechanisms involved in redistribution organelles, including the symbiosomes, clearly merit much more attention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The intracellular infection thread initiated in a root hair cell is a unique structure associated with Rhizobium-legume symbiosis. It is characterized by inverted tip growth of the plant cell wall, resulting in a tunnel that allows invasion of host cells by bacteria during the formation of the nitrogen-fixing root nodule. Regulation of the plant-microbial interface is essential for infection thread growth. This involves targeted deposition of the cell wall and extracellular matrix and tight control of cell wall remodeling. This review describes the potential role of different actors such as transcription factors, receptors, and enzymes in the rearrangement of the plant-microbial interface and control of polar infection thread growth. It also focuses on the composition of the main polymers of the infection thread wall and matrix and the participation of reactive oxygen species (ROS) in the development of the infection thread. Mutant analysis has helped to gain insight into the development of host defense reactions. The available data raise many new questions about the structure, function, and development of infection threads. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Aims Recent findings indicate that Nod factor signalling is tightly interconnected with phytohormonal regulation that affects the development of nodules. Since the mechanisms of this interaction are still far from understood, here the distribution of cytokinin and auxin in pea (Pisum sativum) nodules was investigated. In addition, the effect of certain mutations blocking rhizobial infection and subsequent plant cell and bacteroid differentiation on cytokinin distribution in nodules was analysed. Methods Patterns of cytokinin and auxin in pea nodules were profiled using both responsive genetic constructs and antibodies. Key Results In wild-type nodules, cytokinins were found in the meristem, infection zone and apical part of the nitrogen fixation zone, whereas auxin localization was restricted to the meristem and peripheral tissues. We found significantly altered cytokinin distribution in sym33 and sym40 pea mutants defective in IPD3/CYCLOPS and EFD transcription factors, respectively. In the sym33 mutants impaired in bacterial accommodation and subsequent nodule differentiation, cytokinin localization was mostly limited to the meristem. In addition, we found significantly decreased expression of LOG1 and A-type RR11 as well as KNOX3 and NIN genes in the sym33 mutants, which correlated with low cellular cytokinin levels. In the sym40 mutant, cytokinins were detected in the nodule infection zone but, in contrast to the wild type, they were absent in infection droplets. Conclusions In conclusion, our findings suggest that enhanced cytokinin accumulation during the late stages of symbiosis development may be associated with bacterial penetration into the plant cells and subsequent plant cell and bacteroid differentiation. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]