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).
Two transgenic strains of Rhizobium leguminosarum bv. viciae, 3841-PsMT1 and 3841-PsMT2, were obtained. These strains contain the genetic constructions nifH-PsMT1 and nifH-PsMT2 coding for two pea (Pisum sativum L.) metallothionein genes, PsMT1 and PsMT2, fused with the promoter region of the nifH gene. The ability of both transgenic strains to form nodules on roots of the pea wild-type SGE and the mutant SGECd t , which is characterized by increased tolerance to and accumulation of cadmium (Cd) in plants, was analyzed. Without Cd treatment, the wild type and mutant SGECd t inoculated with R. leguminosarum strains 3841, 3841-PsMT1, or 3841-PsMT2 were similar histologically and in their ultrastructural organization of nodules. Nodules of wild-type SGE inoculated with strain 3841 and exposed to 0.5 μM CdCl 2 were characterized by an enlarged senescence zone. It was in stark contrast to Cd-treated nodules of the mutant SGECd t that maintained their proper organization. Cadmium treatment of either wild-type SGE or mutant SGECd t did not cause significant alterations in histological organization of nodules formed by strains 3841-PsMT1 and 3841-PsMT2. Although some abnormalities were observed at the ultrastructural level, they were less pronounced in the nodules of strain 3841-PsMT1 than in those formed by 3841-PsMT2. Both transgenic strains also differed in their effects on pea plant growth and the Cd and nutrient contents in shoots. In our opinion, combination of Cd-tolerant mutant SGECd t and the strains 3841-PsMT1 or 3841-PsMT2 may be used as an original model for study of Cd tolerance mechanisms in legume-rhizobial symbiosis and possibilities for its application in phytoremediation or phytostabilization technologies.
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.
In Russia, tetramethylthiuram disulfide (TMTD) is a fungicide widely used in the cultivation of legumes, including the pea (Pisum sativum). Application of TMTD can negatively affect nodulation; nevertheless, its effect on the histological and ultrastructural organization of nodules has not previously been investigated. In this study, the effect of TMTD at three concentrations (0.4, 4, and 8 g/kg) on nodule development in three pea genotypes (laboratory lines Sprint-2 and SGE, and cultivar ‘Finale’) was examined. In SGE, TMTD at 0.4 g/kg reduced the nodule number and shoot and root fresh weights. Treatment with TMTD at 8 g/kg changed the nodule color from pink to green, indicative of nodule senescence. Light and transmission electron microscopy analyses revealed negative effects of TMTD on nodule structure in each genotype. ‘Finale’ was the most sensitive cultivar to TMTD and Sprint-2 was the most tolerant. The negative effects of TMTD on nodules included the appearance of a senescence zone, starch accumulation, swelling of cell walls accompanied by a loss of electron density, thickening of the infection thread walls, symbiosome fusion, and bacteroid degradation. These results demonstrate how TMTD adversely affects nodules in the pea and will be useful for developing strategies to optimize fungicide use on legume crops.
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.
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