In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, “locked” infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, “locked” infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.

BACKGROUND: Polyamines are acting as signaling molecules during adaptation to stressful environment and as regulators of plant development. In plants, polyamines are represented mainly by putrescine, spermidine and spermine. The concentration of polyamines in symbiotic nodules of some legumes is 510 times higher than in the other organs, which indicates their important role in the formation and functioning of symbiotic nodules. MATERIALS AND METHODS: We analyzed the expression of genes encoding polyamine biosynthesis enzymes in symbiotic nodules, as well as the effect of exogenous polyamines on the nodule number and the average nodule weight in wild-type SGE plants and symbiotic pea mutants SGEFix-1 (sym40-1) and SGEFix-2 (sym33-3). RESULTS: The comparable expression level of arginine decarboxylase gene (PsADC) was observed in all analyzed nodules, whereas the expression level of ornithine decarboxylase gene (PsODC), was highly increased in nodules of SGEFix-2 (sym33-3) mutant. Treatment of the root system with a 0.1 mM solution of polyamines mixture led to an increase in the average weight of the nodule in wild-type plants and in the SGEFix-2 (sym33-3) mutant plants. CONCLUSIONS: It was shown that the main pathway of putrescine synthesis in wild-type pea symbiotic nodules is the arginine pathway, while the ornithine pathway is probably associated with activation of plant defense reactions. Polyamines acting, apparently, through ethylene, affect the functioning of the nodule meristem.

The garden pea (Pisum sativum L.), like most members of Fabaceae family, is capable of forming symbioses with beneficial soil microorganisms such as nodule bacteria (rhizobia), arbuscular mycorrhizal (AM) fungi and plant growth promoting bacteria (PGPB). The autoregulation of nodulation (AON) system is known to play an important role in controlling both the number of nodules and the level of root colonization by AM via root-to-shoot signaling mediated by CLAVATA/ESR-related (CLE) peptides and their receptors. In the pea, mutations in genes Sym28 (CLV2-like) and Sym29 (CLV1-like), which encode receptors for CLE peptides, lead to the supernodulation phenotype, i.e., excessive nodule formation. The aim of the present study was to analyze the response of pea cv. ‘Frisson’ (wild type) and mutants P64 (sym28) and P88 (sym29) to complex inoculation with rhizobia, AM fungi and PGPB, with regard to biomass accumulation, yield and transcriptomic alterations. The plants were grown in quartz sand for 2 and 4 weeks after inoculation with either rhizobia (Rh) or complex inoculation with Rh + AM, Rh + PGPB, and Rh+AM+PGPB, and the biomass and yield were assessed. Transcriptome sequencing of whole shoots and roots was performed using a modified RNAseq protocol named MACE (Massive Analysis of cDNA Ends). In the experimental conditions, P88 (sym29) plants demonstrated the best biomass accumulation and yield, as compared to the wild type and P64 (sym28) plants, whereas P64 (sym28) had the lowest rate of biomass and seed yield. The transcriptome analysis showed that both supernodulating mutants more actively responded to biotic and abiotic factors than the wild-type plants and demonstrated increased expression of genes characteristic to late stages of nodule development. The roots of P64 (sym28) plants responded to AM+Rh treatment with upregulation of genes encoding plastid proteins, which can be connected with the activation of carotenoid biosynthesis (namely, the non-mevalonate pathway that takes place in root plastids). The more active response to symbionts in P88 (sym29) plants, as compared to cv. ‘Frisson’, was associated with counterregulation of transcripts involved in chloroplast functioning and development in leaves, which accompanies successful plant development in symbiotic conditions. Finally, the effect of retardation of plant aging upon mycorrhization on a transcriptomic level was recorded for cv. ‘Frisson’ but not for P64 (sym28) and P88 (sym29) mutants, which points towards its possible connection with the AON system. The results of this work link the plant’s autoregulation with the responsiveness to inoculation with beneficial soil microorganisms.

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.

Mutualistic symbioses formed by garden pea have been studied with use of ‘omic’ technologies in order to gain a new understanding of molecular mechanisms of beneficial effect that microsymbionts have on seed yield and quality. Keywords: garden pea, transcriptomics, nitrogen fixation, arbuscular mycorrhiza, PGPB

Транскрипционный фактор CYCLOPS/IPD3 является ключевым активатором органогенеза симбиотических клубеньков, он также принимает участие в развитии инфекционных нитей и симбиосом. У гороха было выявлено три мутантные аллели по этому гену ( sym33-1 — sym33-3 ). Наиболее изучены фенотипические проявления аллели sym33-3 у мутанта SGEFix¯-2, характеризующегося «leaky»-фенотипом — формированием двух типов клубеньков: белых и розоватых. Аллель sym33-2 у мутанта SGEFix¯-5 была описана как строгая аллель, тем не менее ее фенотипические проявления не были детально изучены. В данном исследовании проанализирована гистологическая и ультраструктурная организация клубеньков мутанта SGEFix¯-5. В клубеньках наблюдались «запертые» инфекционные нити, из которых не происходил выход бактерий в цитоплазму растительной клетки. При этом в некоторых нитях отмечалась деградация бактерий, что может свидетельствовать об активации сильных защитных реакций в клубеньках мутанта SGEFix¯-5.

2015 (LX) No./Issue 4 The L1 acquisition of differential object marking/ L'acquisition du marquage différentiel de l'objet dans la langue maternelle Édité par / Edited by: Larisa Avram LARISA AVRAM, Editorial: The L1 acquisition of differential object marking [ pdf ] SIGAL UZIEL-KARL, The development of differential object marking in child Hebrew [ pdf ] GORDANA HRžICA, MARIJAN PALMOVI?, MELITA KOVA?EVI?, MARIA D. VOEIKOVA, KIRA IVANOVA, ELENA GALKINA, A nimacy and case in the acquisition of differential object marking in Croatian and Russian [ pdf ] INETA DABAšINSKIEN?, Growing knowledge of differential object marking: The view from L1 Lithuanian [ pdf ] EMMA TICIO, LARISA AVRAM, The acquisition of differential object marking in Spanish and Romanian: Semantic scales or semantic features? [ pdf ]

Metabolic engineering of plant secondary metabolism provides a way to obtain plants with elevated level of valuable molecular compounds. Alternatively, metabolic engineering can be used for reduction of toxic substances accumulation in plant tissues. This approach allows one to expand the application of toxic plants in agriculture and biotechnology. The crops of Solanaceae family provide an example of toxic plants of high economic value. Solanaceae family includes edible crops such as potato, tomato and eggplants, medicinal plants like Withania somnifera L. and major non-food crop Nicotiana tabacum L. The secondary metabolism of Solanaceae family is widely diverse and includes the biosynthesis and accumulation of number of toxic compounds, such as nicotine and other alkaloids in tobacco, steroidal glycoalcaloids in potato and withanolides in winter cherry W. somnifera. The secondary metabolic pathways of Solanaceae family have evolved from primary metabolism via duplication of the enzyme coding genes and diversification of genes functions. Local, segment and the whole genome duplications and subsequent formation of metabolic genes clusters are the main processes in secondary metabolic pathways formation. Recent whole genome sequence data from number of Sonanaceae species allows one to reconstruct the putative mechanism of primary and secondary metabolism genetic control and evolution. Genomic data together with novel guided endonuclease based genome modification tools provide an opportunity for introduction of precise changes into secondary metabolism. Suppression of nicotine accumulation in tobacco is promising approach for developing of novel plant systems for molecular farming. Toxicity of wild potato relatives impedes their usage in potato breeding. Tobacco and wild potato toxicity reduction can be achieved by different genome modification approaches: knock-out of the key enzyme genes of alkaloids synthesis, the large deletion of the whole cluster of the secondary metabolic genes or the precise editing of key transcription factors in secondary metabolism regulation pathways.

Background. The transcription factor CYCLOPS/IPD3 is a key activator of the organogenesis of symbiotic nodules. Its participation in the development of infection threads and symbiosomes is also shown. In pea, three mutant alleles were identified for this gene (sym33-1 sym33-3). The phenotypic manifestations of the sym33-3 allele of the SGEFix-2 mutant, characterized by a leaky phenotype (the formation of two types of nodules: white and pinkish) were the most studied. The sym33-2 allele in the mutant SGEFix-5 was described as a strong allele, however, its phenotypic manifestations have not been studied in detail. Materials and methods. In this study, the histological and ultrastructural nodule organization of the SGEFix-5 mutant was analyzed using confocal laser scanning microscopy and transmission electron microscopy. Results. In the nodules locked infection threads were observed, from which no bacteria release into the cytoplasm of the plant cell occurs. In this case, in some infection threads, bacteria were degraded, which may indicate the activation of strong defense reactions in the nodules of the SGEFix-5 mutant. Conclusions. The sym33-2 allele in the mutant SGEFix-5 is a strong allele, which triggers the severe defense reactions, when rhizobia are already perceived as pathogens in infection threads.

The development of quantitative digital phenotyping methods for evaluation of wild potato (section Petota Dumort., genus Solanum L.) tuberization is required for annotation of genebank collections and selection of the suitable donor material for potato breeding. There are no available methods specifically designed for the quantitative analysis of wild potato tuber morphology. The current study is devoted to evaluation of wild potato tubers’ morphological characteristics using a digital image processing technique. For this purpose, the mobile application SeedSounter developed previously for grain analysis was specifically adapted for tuber phenotyping. The application estimates the number and shape of objects scattered on a standard sheet of white paper (i. e. A3 or A4). Twelve accessions from the VIR genebank collection belonging to nine Petota species were grown in pots protected with garden fabric during the growing season of cultivated potato (Novosibirsk region). Tubers were collected form plants of nine genotypes. Three genotypes did not produce tubers. The weight of tubers collected from each plant was measured. The tuber yield from each plant was analyzed using SeedCounter (http://wheatdb.org/seedcounter). The number of tubers per plant was counted; the following characteristics were extracted from the images of individual tubers: length, width, projected area, length to width ratio, сircularity, roundness, rugosity and solidity. One-way ANOVA showed a significant effect of genotype on all measured characteristics. A pairwise comparison of nine Petota accessions using all measured parameters revealed statistically significant differences between 86 % of pairs. The overall tuber yield volume for each plant was calculated as a sum of volumes of individual tubers; tuber volume was calculated from its length to width ratio and projected area. A strong correlation between the evaluated tuber yield volume and yield weight was shown. We propose tuber yield volume as a characteristic for a general evaluation of tuberization for wild potato, implementing the four-step scale from 0 to 3. According to this characteristic, the twelve wild potato accessions studied could be divided into four groups with different tuberization abilities. The evaluated tuberization ability is partially in accordance with previously obtained VIR data. The results presented demonstrate the possibility to use SeedCounter for wild potato collections phenotyping.

Potato steroidal glycoalkaloids (SGAs) compose a part of plant immunity. Some of their modified variants are toxic to humans. In the course of potato domestication, plants with a lower SGA level were selected. The advent of approaches for manipulation with the regulation of metabolic pathways provides an opportunity to overcome the undesirable direct relationship between the potato resistance to pests and the toxicity of its tubers. However, for such a fine regulation, a deep knowledge of the regulatory network of potato SGA biosynthesis is required. The purpose of this review is to summarize the information on the known SGA biosynthesis genes in plants and the results of the investigation of these genes in potato, as well as to consider the mechanisms of the SGA protective toxic action against pathogens and pests. The SGA biosynthesis is realized via the cytosolic mevalonate pathway and consists of three stages. The first two stages are required for the synthesis of primary metabolites, and lead to cycloartanol and cholesterol, respectively. Twelve enzymes are involved in the biosynthesis, and the half of them are involved in the biosynthesis of phytosterols, which is a branch of the first stage of this metabolic pathway. In the potato leaves with an excess of phytosterols, the synthesis switches to SGAs, increasing the content of the latter. In tubers, with an excess of SGA precursors, they are involved in the synthesis of lanosterol, supporting in this way the stable level of SGA. The importance of structural genes encoding the enzymes of the first two stages of biosynthesis does not allow us to consider them as a target for knockout in order to reduce the level of SGAs. However, information about the tissue-specific mechanisms of switching between the pathways of synthesis of SGA and other compounds having common precursors with SGAs can be used to manipulate the tissue-specific level of steroidal glycoalkaloids. At the third stage (the synthesis of glycoalkaloids from cholesterol), about 20 enzymes participate. In the potato genome, 14 corresponding genes were identified, 8 of which were studied in detail using reverse genetics approaches. As a promising target for reducing SGA levels in tubers, the genes encoding PGA enzymes (belonging to the CYP72 subfamily cytochrome-P450-dependent monooxygenases catalyzing the conversion of hydrocholesterol to trihydrocholesterol) and SGT (SGA glycosyltransferases that catalyze the conversion of solanidine to its toxic glycosylated derivatives α-solanine and α-chaconine) are considered. Cis-regulatory elements in the promoter regions of some glycoalkaloid biosynthesis genes, including elements responsible for tissue-specific expression, are described. The accumulated information provides the base for creating potato genotypes with tissue-specific regulation of SGAs, in which high levels of SGAs in leaves will remain to protect against pathogens and pests and, at the same time, the synthesis of toxic substances in tubers will be suppressed

РОЛЬ НИЗКОМОЛЕКУЛЯРНЫХ ТИОЛОВ В ПРОЦЕССАХ ФОРМИРОВАНИЯ

Circular RNAs (circRNAs) are a new RNA type that show predominantly brain-specific expression pattern in mammals. Their individual representatives, acting as competitive endogenous RNAs, can bind microRNAs and contribute to the protection of functional transcripts. In the model of transient cerebral ischemia in rats, we studied the expression of mGluR 3 and mGluR 5 glutamate metabotropic receptor genes ( Grm3 and Grm5 ). These genes are important participants in the metabolic pathways associated with neurosignaling. In the present study, we found that the rat Grm3 and Grm5 genes, in addition to mRNA, encode circRNAs, which are conserved in humans and rodents. In subcortical brain structures of rats containing a lesion focus, the level of these circRNAs is more stable than that of the corresponding mRNA. Using STarMirDB database analysis, the distribution of the microRNA binding sites along the mRNA molecules of human GRM3 and GRM5 genes which are homologous to the corresponding rat genes was elucidated. It has been revealed that sufficiently large number of binding sites is located within exons, which are also part of conservative circRNAs. The results may indicate the functional role of the circRNAs of the investigated genes as competitive endogenous RNAs in the response of brain cells to ischemia.

Nitrogen-fixing nodules are formed on the roots of leguminous plants as a result of their interaction with soil bacteria, called rhizobia.Nodule development is based on the exchange of signaling molecules that leads to coordinated gene expression in both partners.This process is accompanied by differentiation of both plant and bacterial cells leading to formation of infected plant cells, filled with nitrogen-fixing forms of rhizobia, called bacteroids.The bacteroid is separated from the plant cell cytoplasm by the peribacteroid membrane and forms an organelle-like structure called the symbiosome (A.V. Tsyganova et al., 2017).The main function of the symbiotic nodule is to maintain the microaerophilic conditions required for working of the rhizobial nitrogen fixation enzymenitrogenase, which is extremely sensitive to oxygen.Nitrogen-fixing nodules produce an abundance of reactive oxygen species (ROS) and reactive nitrogen species (RNS).These are formed due to auto-oxidation of leghemoglobin in the cytoplasm, oxidation of nitrogenase and ferredoxin in symbiosomes, and functioning of electron transport chains in mitochondria, symbiosomes, and peroxisomes (C.Chang et al., 2009).ROS and RNS molecules are involved in different signal transduction pathways; therefore, the nodule antioxidant system cannot simply eliminate ROS and RNS, but must maintain their concentration in the cell at the certain level (C.W. Ribeiro et al., 2015).Most antioxidants presented in plant organs are also found in the nodule, however, at a higher concentration, which is probably due to the high intensity of the processes associated with biological nitrogen fixation.These are enzymes superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, and peroxiredoxins, as well as millimolar concentrations of non-enzymatic elements (primarily ascorbic acid and glutathione) (M.Becana et al., 2010).It has been discovered that Legumes harbor a unique homologue of glutathione, homoglutathione, both of which exhibit similar functions and specificity.However, it is still not clear why some Legumes evolved the ability to synthesize two different thiol compounds and require a double regulatory mechanism of the cell cycle including activation by glutathione and inhibition of cytokinesis by homoglutathione (T.Pasternak et al., 2014).It has now been shown that an increase in the level of glutathione leads to an increase in the efficiency of nitrogen fixation, while there is no similar data for homoglutathione.Considering that for the functioning of the nodule a balance in the ratio of glutathione and homoglutathione is necessary, it is evident that increasing the level of nitrogen fixation by modifying the levels of these thiols is a non-trivial task.Moreover, it is necessary to account for the influence of other components of the antioxidant system.It should be noted that the rhizobial antioxidants play an important role in the functioning of the nitrogen fixing nodule (C.W. Ribeiro et al., 2015).In this review, we will consider the main components of the plant antioxidant system in the nodule.A deeper understanding of its functioning is necessary to develop conditions for increasing the efficiency of biological nitrogen fixation.