S. A. Alen’kina

Extracellular Proteolytic Enzymes of Azospirillum brasilense Strain Sp7 and Regulation of Their Activity by a Homologous Lectin

It was found that Azospirillum brasilense strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.

Stabilizing effect of Azospirillum lectins on β-glucosidase activity

Lectins from the surface of Azospirillum brasilense Sp7 and Azospirillum brasilense Sp7.2.3 (a mutant with impaired lectin activity) were shown to induce a stabilizing effect on the activity of almond β-glucosidase under conditions of thermoinactivation and proteolytic enzyme treatment. Differences were revealed in the influence of lectins with various antigenic properties. Our results indicate that the effects of lectins on the catalytic activity of the enzyme are mainly associated with conformational changes in lectin molecules during mutagenesis, but not with carbohydrate specificity (general property). These data should be taken into account in evaluating the role of lectins in the formation of nitrogen-fixing associations.

Comparative assessment of inductive effects of Azospirillum lectins with different antigenic properties on the signal systems of wheat seedling roots

The lectins of associative nitrogen-fixing bacteria Azospirillum brasilense Sp7 and its mutant A. brasilense Sp7.2.3 were shown to have different effects on the components of the wheat seedling root signal system, namely to regulate the levels of cAMP, nitric oxide, diacylglycerol, and salicylic acid, as well as to induce the activities of superoxide dismutase and lipoxygenase. Our results make it possible to consider azospirilla lectins as inducers of the signal systems in wheat seedling roots, since they cause development of several flows of primary signals. These data are of general biological importance, since lectins are present in all living organisms and most of the functions of lectins remain insufficiently understood.

Effect of azospirillum lectins on the activity of proteolytic enzymes and their inhibitors in wheat seedling roots

The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exert a multidirectional effect on the activity of acidic (pH 3.5), neutral (pH 6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.

Change in the content of salicylic acid and activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the influence of Azospirilium lectins

The time course of changes in the endogenous content of salicylic acid, the ratio between the acid’s free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum (A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3) is investigated. Differences in plant response to the action of the lectins from these two strains are established. On the basis of the obtained data, a model is proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in the accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and formation of induced resistance.

Effect of lectins from Azospirillum brasilense to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

A comparative study of the effect of the lectins of Azospirillum brasilense Sp7 and its mutant on the activity of some enzymes in plant cells

Our earlier studies showed that the soil plant-associated nitrogen-fixing bacterium Azospirillum brasilense Sp7 contains a surface glycoprotein (lectin) that is specific to L-fucose (1.87 mM) and D-galactose (20 mM) [1]. The most efficient way to study the function of bacterial surface structures is to use mutants defective in the synthesis of particular components of these structures. However, we did not succeed in obtaining bacterial cells that were unable to synthesize the lectin. On the other hand, we did succeed in obtaining a mutant strain whose cells lost the ability to aggregate erythrocytes and to interact with lectin-specific antibodies but retained surface molecules with a molecular mass and carbohydrate specificity identical to those of the lectin of the parent strain [2]. An understanding of the molecular mechanisms underlying bacterial–plant interactions requires knowledge of the functional role of the lectins of the soil bacteria that form nitrogen-fixing symbioses in the plant rhizosphere. The lectins of azospirilla may perform the role of adhesins that provide for the specific attachment of bacterial cells to wheat roots [3]. Other functions of these lectins are unknown. The aim of this work was to study the ability of the lectins of azospirilla to influence the activity of the hydrolytic enzymes of plant cells.

Azospirillum lectin-induced changes in nitric oxide content in wheat seedling roots

It is shown that lectins of nitrogen-fixing associative bacteria of the genus Azospirillum are able to activate nitric oxide production in wheat seedling roots through the NO signal system and thereby can be inducers of plant adaptation processes.

The Influence of Lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b on the Activity of Plant β-Glucosidase

Previous work from this Lab has shown that the proteins, lectins detected on the surfaces of the associative nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b come into play in interaction with plants at the early stages of establishment of an associative system (Nikitina V.E. et al., 1996). The present work demonstrates the influence of the lectins from A. brasilense Sp7 and A. lipoferum 59b on the activity of β-glucosidase of sweet almonds. Bacteria were grown on synthetic medium (Sadasivan L. et al., 1985) at 37 °C for 18 hours. Agglutinating activity of the cells was estimated with the help of the reaction of hemagglutination, with the use of rabbit erythrocytes treated with trypsin (Lis H. et al., 1972). Isolation of lectins from the cell surface was carried out in accordance with Eshdat (Eshdat Y. et al., 1978). The activity of β-glucosidase was evaluated spectrophotometrically at 425 nm by the amount of the formed nitrophenol (Buglova T.T. et al., 1991). It has been demonstrated that the addition of the increasing concentrations of lectins from A. brasilense Sp7 and A. lipoferum 59b caused the decrease of enzyme activity, hence exerts the inhibiting effect. Maximum inhibiting effect for the lectin from A. brasilense Sp7 was observed at the concentration of 70 mkg/ml (in this case enzyme activity decreased to 85%). The inhibiting effect of the lectin from A. lipoferum 59b was better pronounced (the enzyme activity decreased to 11%) and exhibited at reasonably lower concentrations, namely, 0.32; 0.64; 1.28 mkg/ml.