Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development.

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identif ied these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might inf luence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium inf lux inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1-3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative inf luence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the f luorophores such as red f luorescent protein (RFP) and yellow f luorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by inf iltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modif ication or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

A high coverage reference transcriptome assembly of pea (Pisum sativum L.) mycorrhizal roots.

Arbuscular mycorrhiza (AM) is an ancient mutualistic symbiosis formed by 80-90 % of land plant species with the obligatorily biotrophic fungi that belong to the phylum Glomeromycota. This symbiosis is mutually beneficial, as AM fungi feed on plant photosynthesis products, in turn improving the efficiency of nutrient uptake from the environment. The garden pea (Pisum sativum L.), a widely cultivated crop and an important model for genetics, is capable of forming triple symbiotic systems consisting of the plant, AM fungi and nodule bacteria. As transcriptomic and proteomic approaches are being implemented for studying the mutualistic symbioses of pea, a need for a reference transcriptome of genes expressed under these specific conditions for increasing the resolution and the accuracy of other methods arose. Numerous transcriptome assemblies constructed for pea did not include mycorrhizal roots, hence the aim of the study to construct a reference transcriptome assembly of pea mycorrhizal roots. The combined transcriptome of mycorrhizal roots of Pisum sativum cv. Frisson inoculated with Rhizophagus irregularis BEG144 was investigated, and for both the organisms independent transcriptomes were assembled (coverage 177x for pea and 45x for fungus). Genes specific to mycorrhizal roots were found in the assembly, their expression patterns were examined with qPCR on two pea cultivars, Frisson and Finale. The gene expression depended on the inoculation stage and on the pea cultivar. The investigated genes may serve as markers for early stages of inoculation in genetically diverse pea cultivars.

[Evolution of nitrogen-fixing symbioses based on the migration of bacteria from mycorrhizal fungi and soil into the plant tissues].

The hypothesis is suggested on the emergence of N2-fixing plant symbionts from soil diazotrophs and from the satellites of Glomeromycota fungi forming arbuscular mycorrhizae (AM). This universal form of plant-microbe symbiosis possibly appeared from integration of ancestral land plants (rhyniophytes, psylophytes) and microbial consortia composed of AM-fungi assimilating soil phosphates and bacteria fixing atmospheric CO2 and/or N2. Releasing of these bacteria from AM-fungal hyphae into the plant tissues elicited the selection of genotypes capable of the fungi-independent multiplication in planta, as well as the fixation in bacterial genomes of the genes for synthesis of chitin-like signal factors stimulating the development of symbiotic structures. An early stage of this evolution might been represented by formation of N2-fixing syncyanoses, the late stage - by formation of nodular symbioses of dicots from Eurosid I clade with rhizobia (α- and ß-proteobacteria) and with actinobacteria Frankia. Emergence of these symbioses was possibly based on the migration of soil and endophytic bacteria into the storage organs (modified stems or lateral roots), where the optimal conditions were established not only for N2 fixation but also for the evolution of bacteria towards an increased symbiotic activity. This evolution resulted in the emergence of primary rhizobia (Bradyrhizobium, Burkholderia) which acted as the donors of sym-genes for a broad spectrum of microbes transformed into the secondary rhizobia (Rhizobium, Sinorhizobium). The succeeding evolution of nodular symbioses was directed at an increased efficiency of symbiotrophic nitrogen nutrition in host plants following two scenarios: (i) "expensive", based on the increase of N2- fixing activity via transformation of bacteria into non-reproducible bacteroids; (ii) "economic", based on acquiring the determinate nodule structure and ureide nitrogen assimilation.

[The Principle of Genome Complementarity in the Enhancement of Plant Adaptive Capacities].

In the present work, the potential for the enhancement of the adaptive capacity of microbe-plant systems (MPSs) through the integration of the symbiosis partners' genomes is considered on the example of different types of symbiotic relationships. The accumulated data on the genetic control of interactions for both the plant and microbe, which are discussed in the paper with respect to signaling genes, suggest that it is the complementarity of genetic determinants that underlies the successful formation of MPSs. A eukaryotic genome with limited information content, which is stable throughout a generation, is complemented by a virtually unlimited prokaryotic metagenome. The microsymbiont's ability to adapt to different living conditions is based on the restructuring of the accessory genome by different mechanisms, which are likely to be activated under the influence of plants, although the details of such a regulation remain unknown. Features of the genetic control of the interaction, particularly its universal character for different symbionts, allow us to formulate a principle of genome-complementarity with respect to interacting organisms and consider it an important factor, an adaptation that enhances the abilities of M PSs for their sustainable development in natural ecosystems and for high plant productivity in agrocenoses.

KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule development.

KNOX transcription factors (TFs) regulate different aspects of plant development essentially through their effects on phytohormone metabolism. In particular, KNOX TF SHOOTMERISTEMLESS activates the cytokinin biosynthesis ISOPENTENYL TRANSFERASE (IPT) genes in the shoot apical meristem. However, the role of KNOX TFs in symbiotic nodule development and their possible effects on phytohormone metabolism during nodulation have not been studied to date. Cytokinin is a well-known regulator of nodule development, playing the key role in the regulation of cell division during nodule primordium formation. Recently, the activation of IPT genes was shown to take place during nodulation. Therefore, it was hypothesized that KNOX TFs may regulate nodule development and activate cytokinin biosynthesis upon nodulation. This study analysed the expression of different KNOX genes in Medicago truncatula Gaertn. and Pisum sativum L. Among them, the KNOX3 gene was upregulated in response to rhizobial inoculation in both species. pKNOX3::GUS activity was observed in developing nodule primordium. KNOX3 ectopic expression caused the formation of nodule-like structures on transgenic root without bacterial inoculation, a phenotype similar to one described previously for legumes with constitutive activation of the cytokinin receptor. Furthermore, in transgenic roots with MtKNOX3 knockdown, downregulation of A-type cytokinin response genes was found, as well as the MtIPT3 and LONELYGUY2 (MtLOG2) gene being involved in cytokinin activation. Taken together, these findings suggest that KNOX3 gene is involved in symbiotic nodule development and may regulate cytokinin biosynthesis/activation upon nodule development in legume plants.

[Features of the expression of a meristem-specific WOX5 gene during nodule organogenesis in legumes].

In recent years, the role of WOX genes encoding homeodomain transcription factors in the development of the apical meristem of shoots and roots has been actively investigated. However, the role of WOX genes in the control of the cell proliferation in other meristem types is poorly studied. In our work, we have studied the role of the WOX5 gene in the development of the meristem in nitrogen-fixing nodules developing on the roots of legumes in a symbiosis with rhizobia. We have shown that the WOX5 gene is involved in the development of the nodule meristem in legumes, have quantitatively evaluated the gene's expression at different nodule formation stages, and have studied the localization of its expression using a construction containing the WOX5 promoter conjugated with a reporter gene. The role of the WOX5 transcription factor in the nodule organogenesis and its possible interaction with the hormonal system in the course of the nodule development has been discussed.

Genetic dissection of Rhizobium-induced infection and nodule organogenesis in pea based on ENOD12A and ENOD5 expression analysis.

In legumes, perception of rhizobial lipochitooligosacharide-based molecules (Nod factors) and subsequent signal transduction triggers transcription of plant symbiosis-specific genes (early nodulins). We present genetic dissection of Nod factor-controlled processes in Pisum sativum using two early nodulin genes PsENOD12a and PsENOD5, that are differentially up-regulated during symbiosis. A novel set of non-nodulating pea mutants in fourteen loci was examined, among which seven loci are not described in Lotus japonicus and Medicago truncatula. Mutants defective in Pssym10, Pssym8, Pssym19, Pssym9 and Pssym7 exhibited no PsENOD12a and PsENOD5 activation in response to Nod factor-producing rhizobia. Thus, a conserved signalling module from the LysM receptor kinase encoded by Pssym10 down to the GRAS transcription factor encoded by Pssym7 is essential for Nod factor-induced gene expression. Of the two investigated genes, PsENOD5 was more strictly regulated; not only requiring the SYM10-SYM7 module, but also SYM35 (NIN transcription factor), SYM14, SYM16 and SYM34. Since Pssym35, Pssym14, Pssym34 and Pssym16 mutants show arrested infection and nodule formation at various stages, PsENOD5 expression seems to be essential for later symbiotic events, when rhizobia enter into plant tissues. Activation of PsENOD12a only requires components involved in early steps of signalling and can be considered as a marker of early symbiotic events preceding infection.

[Investigation of systemic control of plant cell division and differentiation in the model of tumor growth in radish].

The study addresses the control of plant cell division and differentiation using the model of tumor-forming lines of radish. Expression of the genes involved in control of the cell cycle (CycD3), maintenance of meristematic cell activity (STM, WUS, and KNAT1), and primary response to cytokinin (ARR) was studied in inbred radish lines characterized by tumor growth at different stages of development. The influence of exogenic cytokinin on the expression of the genes of interest is analyzed. The possible role of the CycD3, KNAT1, STM, WUS, and ARR5 in tumor formation in radish is discussed.

[Metabolic integration of organisms within symbiotic systems].

Adaptation of organisms to coexisence in symbiotic systems is usually related to significant metabolic changes resulting in the integration of the biochemical pathways of the partners. In the symbioses between plants and nitrogen-fixing organisms, between heterotrophic and autotrophic organisms, as well as between animals and microorganisms providing the consumption of plant biomass, the systems of C- and N-metabolism, controlling the utilization of various sources of nitrogen (N2, organic and inorganic compounds, metabolic waste of the host) and carbon (CO2, plant polymers), of the partners are tightly integrated. Bilateral biochemical links between partners are typical to mutualistic symbioses (wherein biotrophic nutrition predominates, in some cases including necrotrophy of secondary origin). In antagonistic symbioses, unilateral links predominate, though active assimilation of the pathogen's secondary metabolites by the host is also possible. In most mutualistic symbioses, integrated metabolic ties have derived from trophic chains in biocenoses (syntrophic consortia, "predator-prey" systems), but not from the systems where the pathogens consume host metabolites. At the same time, molecular analysis of symbiotic interactions has shown that symbioses considerably differ from biocenoses, where the cycling of nutrients and energy implies no functional integration of the partner's genes.

[Lactate dehydrogenase activity and changes in its isoenzyme spectrum in the liver of the bream Abramis brama parasitized by pleurocercoids of the cestode Digramma interrupta]. / Aktivnost' laktatdegidrogenazy i izmenenie spektra ee izofermentov v pecheni leshcha Abramis brama pri parazitirovanii plerotserkoidov tsestody Digramma interrupta.

Changes of lactate-dehydrogenase (KF 1.1.1.27) activity have been studied in the liver of the bream been infected with pleurocercoids of the cestode Digramma interrupta. It is showed, that an increase of enzyme activity depends upon a slowly migrating from LDH-6. Problems of interaction at the level of substrate within a system parasite-host is considered.

[An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae]. / Izuchenie fermentov biosinteza purinovykh nukleotidov u plevrotserkoidov tsestod sem. Ligulidae.

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.