Effects of Elevated Temperature on Pisum sativum Nodule Development: II-Phytohormonal Responses.

High temperature is one of the most important factors limiting legume productivity. We have previously shown the induction of senescence in the apical part of nodules of the pea SGE line, formed by Rhizobium leguminosarum bv. viciae strain 3841, when they were exposed to elevated temperature (28 °C). In this study, we analyzed the potential involvement of abscisic acid (ABA), ethylene, and gibberellins in apical senescence in pea nodules under elevated temperature. Immunolocalization revealed an increase in ABA and 1-aminocyclopropane-1-carboxylic acid (ACC, the precursor of ethylene biosynthesis) levels in cells of the nitrogen fixation zone in heat-stressed nodules in 1 day of exposure compared to heat-unstressed nodules. Both ABA and ethylene appear to be involved in the earliest responses of nodules to heat stress. A decrease in the gibberellic acid (GA3) level in heat-stressed nodules was observed. Exogenous GA3 treatment induced a delay in the degradation of the nitrogen fixation zone in heat-stressed nodules. At the same time, a decrease in the expression level of many genes associated with nodule senescence, heat shock, and defense responses in pea nodules treated with GA3 at an elevated temperature was detected. Therefore, apical senescence in heat-stressed nodules is regulated by phytohormones in a manner similar to natural senescence. Gibberellins can be considered as negative regulators, while ABA and ethylene can be considered positive regulators.

Effects of Elevated Temperature on Pisum sativum Nodule Development: I-Detailed Characteristic of Unusual Apical Senescence.

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.

Rhizobacteria Mitigate the Negative Effect of Aluminum on Pea Growth by Immobilizing the Toxicant and Modulating Root Exudation.

High soil acidity is one of the main unfavorable soil factors that inhibit the growth and mineral nutrition of plants. This is largely due to the toxicity of aluminum (Al), the mobility of which increases significantly in acidic soils. Symbiotic microorganisms have a wide range of beneficial properties for plants, protecting them against abiotic stress factors. This report describes the mechanisms of positive effects of plant growth-promoting rhizobacteria Pseudomonas fluorescens SPB2137 on four pea (Pisum sativum L.) genotypes grown in hydroponics and treated with 80 µM AlCl3. In batch culture, the bacteria produced auxins, possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, alkalized the medium and immobilized Al, forming biofilm-like structures and insoluble phosphates. Inoculation with Ps. fluorescens SPB2137 increased root and/or shoot biomass of Al-treated plants. The bacteria alkalized the nutrient solution and transferred Al from the solution to the residue, which contained phosphorus that was exuded by roots. As a result, the Al concentration in roots decreased, while the amount of precipitated Al correlated negatively with its concentration in the solution, positively with the solution pH and negatively with Al concentration in roots and shoots. Treatment with Al induced root exudation of organic acids, amino acids and sugars. The bacteria modulated root exudation via utilization and/or stimulation processes. The effects of Al and bacteria on plants varied depending on pea genotype, but all the effects had a positive direction and the variability was mostly quantitative. Thus, Ps. fluorescens SPB2137 improved the Al tolerance of pea due to immobilization and exclusion of toxicants from the root zone.

Tubulin Cytoskeleton Organization in Cells of Determinate Nodules.

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.

General Patterns and Species-Specific Differences in the Organization of the Tubulin Cytoskeleton in Indeterminate Nodules of Three Legumes.

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).

Mutational analysis indicates that abnormalities in rhizobial infection and subsequent plant cell and bacteroid differentiation in pea (Pisum sativum) nodules coincide with abnormal cytokinin responses and localization.

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.

Efficacy of a Plant-Microbe System: Pisum sativum (L.) Cadmium-Tolerant Mutant and Rhizobium leguminosarum Strains, Expressing Pea Metallothionein Genes PsMT1 and PsMT2, for Cadmium Phytoremediation.

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 SGECdt, which is characterized by increased tolerance to and accumulation of cadmium (Cd) in plants, was analyzed. Without Cd treatment, the wild type and mutant SGECdt 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 CdCl2 were characterized by an enlarged senescence zone. It was in stark contrast to Cd-treated nodules of the mutant SGECdt that maintained their proper organization. Cadmium treatment of either wild-type SGE or mutant SGECdt 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 SGECdt 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.

Mapping-by-sequencing using NGS-based 3'-MACE-Seq reveals a new mutant allele of the essential nodulation gene Sym33 (IPD3) in pea (Pisum sativum L.).

Large collections of pea symbiotic mutants were accumulated in the 1990s, but the causal genes for a large portion of the mutations are still not identified due to the complexity of the task. We applied a Mapping-by-Sequencing approach including Bulk Segregant Analysis and Massive Analysis of cDNA Ends (MACE-Seq) sequencing technology for genetic mapping the Sym11 gene of pea which controls the formation of symbioses with both nodule bacteria and arbuscular-mycorrhizal fungi. For mapping we developed an F 2-population from the cross between pea line N24 carrying the mutant allele of sym11 and the wild type NGB1238 (=JI0073) line. Sequencing libraries were prepared from bulks of 20 plants with mutant and 12 with wild-type phenotype. MACE-Seq differential gene expression analysis between mutant-phenotype and wild-type-phenotype bulks revealed 2,235 genes, of which 514 (23%) were up-regulated and 1,721 (77%) were down-regulated in plant roots inoculated with rhizobia as a consequence of sym11 mutation. MACE-Seq also detected single nucleotide variants between bulks in 217 pea genes. Using a novel mathematical model we calculated the recombination frequency (RF) between the Sym11 gene and these 217 polymorphic genes. Six genes with the lowest RF were converted into CAPS or dCAPS markers and genetically mapped on the complete mapping population of 108 F 2-plants which confirmed their tight linkage to Sym11 and to each other. The Medicago truncatula Gaertn. (Mt) homologs of these genes are located in a distinct region of Mt chromosome 5, which corresponds to linkage group I of pea. Among 94 candidate genes from this region only one was down-regulated-the pea Sym33 homolog of the Mt IPD3 gene which is essential for nodulation. Sequencing of the Sym33 allele of the N24 (sym11) mutant revealed a single nucleotide deletion (c.C319del) in its third exon resulting in a codon shift in the open reading frame and premature translation termination. Thus, we identified a novel mutant allele sym33-4 most probably responsible for the mutant phenotype of the N24 (sym11) line, thereby demonstrating that mapping by MACE-Seq can be successfully used for genetic mapping of mutations and identification of candidate genes in pea.

Structural Variations in LysM Domains of LysM-RLK PsK1 May Result in a Different Effect on Pea⁻Rhizobial Symbiosis Development.

Lysin-motif receptor-like kinase PsK1 is involved in symbiosis initiation and the maintenance of infection thread (IT) growth and bacterial release in pea. We verified PsK1 specificity in relation to the Nod factor structure using k1 and rhizobial mutants. Inoculation with nodO and nodE nodO mutants significantly reduced root hair deformations, curling, and the number of ITs in k1-1 and k1-2 mutants. These results indicated that PsK1 function may depend on Nod factor structures. PsK1 with replacement in kinase domain and PsSYM10 co-production in Nicotiana benthamiana leaves did not induce a hypersensitive response (HR) because of the impossibility of signal transduction into the cell. Replacement of P169S in LysM3 domain of PsK1 disturbed the extracellular domain (ECD) interaction with PsSYM10's ECD in Y2H system and reduced HR during the co-production of full-length PsK1 and PsSYM0 in N. benthamiana. Lastly, we explored the role of PsK1 in symbiosis with arbuscular mycorrhizal (AM) fungi; no significant differences between wild-type plants and k1 mutants were found, suggesting a specific role of PsK1 in legume⁻rhizobial symbiosis. However, increased sensitivity to a highly aggressive Fusarium culmorum strain was found in k1 mutants compared with the wild type, which requires the further study of the role of PsK1 in immune response regulation.

Cell differentiation in nitrogen-fixing nodules hosting symbiosomes.

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. We will also consider differentiation of symbiosomes, paying attention to the role of NCR peptides, 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.

Comparative analysis of the tubulin cytoskeleton organization in nodules of Medicago truncatula and Pisum sativum: bacterial release and bacteroid positioning correlate with characteristic microtubule rearrangements.

In this study we analyzed and compared the organization of the tubulin cytoskeleton in nodules of Medicago truncatula and Pisum sativum. We combined antibody labeling and green fluorescent protein tagging with laser confocal microscopy to observe microtubules (MTs) in nodules of both wild-type (WT) plants and symbiotic plant mutants blocked at different steps of nodule development. The 3D MT organization of each histological nodule zone in both M. truncatula and P. sativum is correlated to specific developmental processes. Endoplasmic MTs appear to support infection thread growth, infection droplet formation and bacterial release into the host cytoplasm in nodules of both species. No differences in the organization of the MT cytoskeleton between WT and bacterial release mutants were apparent, suggesting both that the phenotype is not linked to a defect in MT organization and that the growth of hypertrophied infection threads is supported by MTs. Strikingly, bacterial release coincides with a change in the organization of cortical MTs from parallel arrays into an irregular, crisscross arrangement. After release, the organization of endoplasmic MTs is linked to the distribution of symbiosomes. The 3D MT organization of each nodule histological zone in M. truncatula and P. sativum was analyzed and linked to specific developmental processes.