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Applied Biochemistry and Microbiology (v.43, #3)

To the hundredth anniversary of the birth of Vaclaw Leonovich Kretovich by A. F. Topunov (pp. 233-236).

Regulatory genes of garden pea (Pisum sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: A review of basic and applied aspects by A. Yu. Borisov; T. N. Danilova; T. A. Koroleva; E. V. Kuznetsova; L. Madsen; M. Mofett; T. S. Naumkina; T. A. Nemankin; E. S. Ovchinnikova; Z. B. Pavlova; N. E. Petrova; A. G. Pinaev; S. Radutoiu; S. M. Rozov; T. S. Rychagova; O. Yu. Shtark; I. I. Solovov; J. Stougaard; I. A. Tikhonovich; A. F. Topunov; V. E. Tsyganov; A. G. Vasil’chikov; V. A. Voroshilova; N. F. Weeden; A. I. Zhernakov; V. A. Zhukov (pp. 237-243).

The review sums up the long experience of the authors and other researchers in studying the genetic system of garden pea (Pisum sativum L.), which controls the development of nitrogen-fixing symbiosis and arbuscular mycorrhiza. A justified phenotypic classification of pea mutants is presented. Progress in identifying and cloning symbiotic genes is adequately reflected. The feasibility of using double inoculation as a means of increasing the plant productivity is demonstrated, in which the potential of a tripartite symbiotic system (pea plants-root nodule bacteria-arbuscular mycorrhiza) is mobilized.

Glutamine synthetase of the rhizobacterium Azospirillum brasilense: Specific features of catalysis and regulation by L. P. Antonyuk (pp. 244-249).

Data on glutamine synthetase (GS) of Azospirillum brasilense, a plant growth-promoting rhizobacterium, have been reviewed. GS of the azospirillum is a type α₁₂ dodecamer with oligomer and monomer having molecular weights of 630 and 52 kDa, respectively. Glutamine synthesis is performed in 12 active sites of the enzyme, depending, first and foremost, on the extent of GS adenylylation and, secondarily, on the exact bivalent metal cations involved in the catalysis. Structural characteristics and catalytic properties of the completely unadenylylated and moderately adenylated forms of GS of A. brasilense have been studied. The enzyme appears as a highly structured protein, with α helices and β structures accounting for about 70% of the polypeptide chain length. Binding of Mg²⁺, Co²⁺, and Mn²⁺ to the protein globule changes both the secondary structure and the catalytic properties of the enzyme. The use of nuclear gamma resonance emission spectroscopy demonstrates that the active center of GS of the azospirillum has two metal-binding sites differing in their affinity for Co²⁺. The activity and biosynthesis of GS of the azospirillum is regulated by wheat lectin (a molecular signal of the host plant), in addition to other means of regulation described for GSs.

Nitrogen-fixing cyanobacteria: A review by A. A. Tsygankov (pp. 250-259).

The review sums up recent data on the key enzymes involved in hydrogen evolution. Hydrogen release by cyanobacteria is due to the effects of nitrogenases and hydrogenases. Cyanobacteria hold important practical implications as transformers of solar energy into molecular hydrogen viewed as a fuel. Avenues of further research in the field are discussed.

The defense and regulatory mechanisms during development of legume—Rhizobium symbiosis by A. K. Glyan’ko; G. P. Akimova; M. G. Sokolova; L. E. Makarova; G. G. Vasil’eva (pp. 260-267).

The roles of indolylacetic acid, the peroxidase system, catalase, active oxygen species, and phenolic compounds in the physiological and biochemical mechanisms involved in the autoregulation of nodulation in the developing legume—Rhizobium symbiosis were studied. It was inferred that the concentration of indolylacetic acid in the roots of inoculated plants, controlled by the enzymes of the peroxidase complex, is the signal permitting or limiting nodulation at the initial stages of symbiotic interaction. Presumably, the change in the level of active oxygen species is determined by an antioxidant activity of phenolic compounds. During the development of symbiosis, phytohormones, antioxidant enzymes, and active oxygen species may be involved in the regulation of infection via both a direct antibacterial action and regulation of functional activity of the host plant defense systems.

The effect of inoculation with Rhizobium leguminosarum on the contents of cytoplasmic protein and free amino acids in the roots of pea seedlings by M. G. Sokolova; G. P. Akimova; L. V. Nechaeva; A. V. Permyakov; A. M. Sobenin (pp. 268-273).

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root zone (0–5 mm) and decreased in the root zones susceptible to rhizobia (5–20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.

Efficacy of biological preparations of soybean root nodule bacteria modified with a homologous lectin by D. M. Sytnikov; S. Ya. Kots’; V. K. Datsenko (pp. 274-279).

The effect of various biopreparations of the root nodule bacterium Bradyrhizobium japonicum, modified with a homologous lectin, on the virulence of rhizobia, the nitrogen-fixing activity of root nodules, and the productivity of the soybean (Glycine max (L.) Merr.) was studied. It was shown that a homologous lectin, added to a bacterial suspension when manufacturing biopreparations on a liquid and solid support, increases the efficiency of the soybean symbiotic system and the productivity of the host plant. The potentialities of using bacterial preparations modified with a homologous lectin are discussed.

The symbiosis between oriental goat’s rue and the root nodule bacteria Rhizobium galegae: Specificity and competitiveness by Al. Kh. Baimiev; I. I. Gubaidullin; An. Kh. Baimiev; A. V. Chemeris; Kh. M. Baimiev; V. A. Vakhitov (pp. 280-285).

Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat’s rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat’s rue or had adapted to infecting this species due to various genetic rearrangements.

The effect of combined and separate inoculation of alfalfa plants with Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and nitrogen-fixing activities by E. K. Furina; G. A. Bonartseva (pp. 286-291).

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N₂O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N₂O with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C₂H₄ nmol/flask · 24 h) and low denitrification activity (1.8 μmol N₂O/flask · 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.

The effect of metabolites on the pH gradient and membrane potential of the bean peribacteroid membrane by V. V. Krylova; N. P. Dubrovo; S. F. Izmailov (pp. 292-297).

The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (ΔpH) and membrane potential (Δψ) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of ΔpH in PBM of young developing nodules in the presence of the K⁺/H⁺ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased ΔpH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both ΔpH in the presence of nigericin in the medium and Δψ generated on PBM by H⁺-ATPase. A decrease in the effects of metabolites on ΔpH and the absent activity of the PBM H⁺ pump were observed in the aging nodules. The obtained data on the changes in ΔpH and Δψ caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.

A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities by S. Yu. Filippovich; G. P. Bachurina; M. S. Kritsky (pp. 298-303).

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.

Physiological and biochemical properties of the bacterial association of Klebsiella terrigena E6 and Bacillus firmus E3 by A. K. Zlotnikova; M. L. Kazakova; K. M. Zlotnikov; A. V. Kazakov; M. M. Umarov (pp. 304-312).

Physiological and biochemical properties of the natural rhizospheric association of Klebsiella terrigena E6 and Bacillus firmus E3 were studied. It was demonstrated that this association fixed actively molecular nitrogen at a rate of 110 nmol ethylene per 1 mg protein per h. The dynamics of bacterial population in the association and in pure cultures were studied. The properties of this association depended on the ratio of its components; the maximal nitrogen fixation was recorded at a content of B. firmus E310 amounting to 15% of the total cell number. It was demonstrated that the stability basis of this association was the specific interactions via its components via metabolites—phenol and para-hydroxybenzoic acid—as well as via nitrogen fixation, respiration, and pH of the medium. A scheme of the interactions between the components of association is shown.

Method of determination of the content of hemoglobin-like proteins in heterogenic mixtures by O. V. Kosmachevskaya; A. F. Topunov (pp. 313-319).

A spectrophotometric method of quantification of hemoglobin-like proteins, which makes it possible to determine their concentration in mixtures of various composition, was proposed. This method is based on comparing the optical density of different redox forms of proteins when their spectra are superposed at the isosbestic point. The coefficients used in calculations were determined by us. The proposed technique is simple and does not require special procedures. It was tested using myoglobin solutions of various concentrations, myoglobin solutions containing other proteins, and solutions of leghemoglobin (a myoglobin-like protein from legumes).

A rapid method to evaluate potential vasodilatory activity of organic nitrates by L. A. Syrtsova; B. L. Psikha; E. S. Malkova; N. I. Shkondina; A. I. Kotel’nikov (pp. 320-323).

The kinetics of interaction between organic nitrates (3,3-bis(nitroxymethyl)oxetane) and cysteine were evaluated by the rate of nitrite ion formation at various concentrations of reagents and pH. The activities of natural reducing agents, including cysteine, glutathione, and NADH, in generating the nitrite ion from organic nitrates (3,3-bis(nitroxymethyl)oxetane) were compared. Cysteine was shown to be the most potent reducing agent. Studying the effectiveness of nitrates (trinitroglycerol, 3,3-bis(nitroxymethyl)oxetane, and nicorandil) at a concentration of 3 mM showed that the rate of nitrite ion accumulation in the reaction with 10 mM cysteine is 1.66, 0.37, and 0.02 μM/min, respectively. The reaction of organic nitrate with cysteine (Cys) is used as a test system for analyzing the effectiveness of nitrates in nitrite ion formation, which correlates with vasodilatory activity of these compounds (dilation of blood vessels).

Biochemical aspects of growth-stimulating effects of steroid phytohormones on lupine plants by O. L. Kandelinskaya; A. F. Topunov; E. R. Grishchenko (pp. 324-331).

Presowing treatment of seeds of lupine of various species and cultivars with brassinosteroids—homobrassinolide and epibrassinolide—caused an increase in protein content and a change in the proportion of some amino acids. Both hormones increased heterogeneity of high-and medium-molecular-weight nonhistone chromatin proteins but had no effect on the polypeptide profile of histones. Using epibrassinolide as an example, we showed that the brassinosteroid-induced increase in protein content in lupine seeds was due primarily to accumulation of the low-molecular-weight components of β-conglutin. The content of some amino acids in both α-and β-conglutins changed. These changes in protein metabolism correlated with an increase in the content of indoleacetic acid and a decrease in the content of abscisic acid.

Physiological and biochemical characteristics of the recalcitrant seeds having dormancy: A review by N. A. Gumilevskaya; M. I. Azarkovich (pp. 332-340).

The review considers and sums up the results of studies of physiological and biochemical characteristics of the dormant and germinating recalcitrant seed (the object of the study, the seed of horse chestnut, Aesculus hippocastanum L., is viewed as an exemplary case). The results of analysis of the proteomes of the axes and cotyledons have been studied and the effects of the stratification have been assessed. Gene expression has been studied at the level of protein synthesis: the protein-synthesizing capacity of the cells of the embryonic axis and cotyledon storage parenchyma of mature seed and seed undergoing stratification. The extent to which the functionally active translation machinery of ripe seed depends on transcription has been assessed, and the ability to synthesize protein under the conditions of stratification has been established. It is concluded that the embryonic axis of dormant seed lacks innate dormancy and that the isolated axis exhibits diverse sensitivity to exogenous abscisic acid and other physiologically active compounds.

Role of transamination in the mobilization of respiratory substrates in germinating seeds of castor oil plants by V. N. Popov; A. T. Eprintsev; D. N. Fedorin; O. Yu. Fomenko; A. U. Igamberdiev (pp. 341-346).

The metabolism of 1,4-¹⁴C-succinate and 2,3-¹⁴C-succinate and the activity of succinic semialdehyde dehydrogenase (EC 1.2.1.16) were studied in germinating seeds of castor oil plants (Ricinus communis L.). Succinate metabolism involved succinate dehydrogenase and was sensitive to metabolites of the γ-aminobutyric acid shunt. Considerable accumulation of the label in amino acids reflected the progression of transamination reactions. Succinic semialdehyde dehydrogenase was purified from the endosperm of castor oil plants. Kinetic characteristics of the enzyme were evaluated. Our study indicates that the mobilization of respiratory substrates during germination of castor oil plants is related to active transamination of ketoacids in the Krebs cycle and involves the γ-aminobutyric acid shunt.

Physicochemical properties of proteins from wheat grown under high-contrast climatic conditions by V. V. Kolpakova; E. N. Molchanova; A. V. Vasil’ev; L. V. Chumikina (pp. 347-355).

Studies using electrophoresis, gel chromatography, viscometry, and calorimetry revealed an interrelation of several physicochemical properties of proteins of soft wheat grown under conditions of cool and wet weather with rheological characteristics of gluten and dough and bread quality. The ratio of gliadin and albumin-globulin polypeptides in flour with short-tearing gluten was much lower compared to that in flour with normal gluten. Proteins from flour with short-tearing gluten, including the water-soluble and salt-soluble fraction, had a loose spatial structure. Gluten fractions of this gluten (gliadin and glutenin) were characterized by a more compact and elongated structure compared to normal gluten. As distinct from normal gluten, the conformation of protein particles in short-tearing gluten depended little on hydrophobic interactions. The results suggest that the main components of grain determine the rheological properties of short-tearing gluten.

The protein-proteinase complex of the barley grown on a different agronomic background using regulatory preparations by I. S. Vitol; G. P. Karpilenko (pp. 356-364).

An integrated effect of agronomic background (the degree of soil fertility and the dose of applied fertilizers) and the regulatory preparations of phenolic and terpene natures on the protein-proteinase complex of brewer’s barley grown under conditions of the nonchernozem zone was studied. Gel chromatography methods were used to demonstrate that the treatment with preparations of regulatory substances increased the degree and depth of storage protein hydrolysis during malting and provided the necessary degree of hydrolysis of the endosperm proteins, an important characteristic of the ready malt. Neutral proteinases hydrolyzed proteins forming intermediate products with various molecular weights, thereby indicating their sufficiently narrow specificity. Acid proteinases generated a large amount of low-molecular-weight products, confirming a wide specificity of their action and their ability to hydrolyze the peptide bonds formed by various amino acid residues.

“Laccase of the lignolytic fungus Trametes hirsuta: Purification and characterization of the enzyme and cloning and primary structure of the gene” by D. V. Rebrikov; E. V. Stepanova; O. V. Koroleva; Zh. I. Budarina; M. V. Zakharova; T. V. Yurkova; A. S. Solonin; O. V. Belova; Z. A. Pozhidaeva; A. A. Leont’evsky (pp. 365-365).

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Applied Biochemistry and Microbiology (v.43, #3)

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Applied Biochemistry and Microbiology (v.43, #3)