ABSCISIC ACID AND ETHYLENE PRODUCTION BY BIOTECHNOLOGICAL STRAINS OF Bradyrhizobium japonicum

64 Due to the environmental impact caused by people, which threatens increasingly agriculture of Ukraine, there is a need to restore natural ecosystems, to maintain their biological diversity and to protect them from destruction. Therefore, it is important to create and apply modern biotechnologies based on the use of environmentally safe microbiological preparations of new generation with combined properties of means for increasing crop yields and their resistance to extreme environmental factors. In recent years efforts of scientists were focused on development of complex bacterial preparations (inoculants) based on living nitrogenfixing soil microorganisms, which include phytohormones, vitamins, amino acids and other physiologically active substances. At establishing symbiotic interactions between nodule bacteria and leguminous plants the highly specialized structures — nodules are formed which develop on host plants roots, and in which the nitrogen fixation takes place directly [1]. Mechanism of nodules formation (or nodulation) is highly specific. It includes cascade of chemical reactions with signaling molecules of different chemical nature. Among them the phytohormones of both the plants and bacterial origin are important (auxins, cytokinins, gibberellins, abscisic acid and ethylene). The ability to production phytohormones is inherent in a wide range of soil microorganisms. Among them there are the symbiotic nitrogen–fixing bacteria (rhizobia, from families Rhizobiaceae and Bradyrhizobiaceae) [2–4]. It is known that the phytohormones synthesized by rhizobia, take part in regulation of such processes of symbiosis as: rhizogenes stimulation; increase of roots surface area; enhancement of exometabolites exchange efficiency in the plant–soil–organisms system; stimulation of plant roots cell proliferation (nodulation); control of nodules number and they mass; increase nitrogenase activity of bacteroides; enhancement of plant resistance to environmental stress factors [5–7]. Previously we have researched biosynthesis of the phytohormones auxins and cytokinins by free–living bacteria Azotobacter chroococcum and symbiotic strains of Bradyrhizobium japonicum with various effectiveness rates [8, 9]. However, the ability of these UDC 579.222.3 + 577.175.15 + 631.847.211 doi: 10.15407/biotech8.05.064

Due to the environmental impact caused by people, which threatens increasingly agriculture of Ukraine, there is a need to restore natural ecosystems, to maintain their biological diversity and to protect them from destruction.Therefore, it is important to create and apply modern biotechnologies based on the use of environmentally safe microbiological preparations of new generation with combined properties of means for increasing crop yields and their resistance to extreme environmental factors.In recent years efforts of scientists were focused on development of complex bacterial preparations (inoculants) based on living nitrogenfixing soil microorganisms, which include phytohormones, vitamins, amino acids and other physiologically active substances.
At establishing symbiotic interactions between nodule bacteria and leguminous plants the highly specialized structures -nodules are formed which develop on host plants roots, and in which the nitrogen fixation takes place directly [1].Mechanism of nodules formation (or nodulation) is highly specific.It includes cascade of chemical reactions with signaling molecules of different chemical nature.Among them the phytohormones of both the plants and bacterial origin are important (auxins, cytokinins, gibberellins, abscisic acid and ethylene).
The ability to production phytohormones is inherent in a wide range of soil microorganisms.Among them there are the symbiotic nitrogen-fixing bacteria (rhizobia, from families Rhizobiaceae and Bradyrhizobiaceae) [2][3][4].It is known that the phytohormones synthesized by rhizobia, take part in regulation of such processes of symbiosis as: rhizogenes stimulation; increase of roots surface area; enhancement of exometabolites exchange efficiency in the plant-soil-organisms system; stimulation of plant roots cell proliferation (nodulation); control of nodules number and they mass; increase nitrogenase activity of bacteroides; enhancement of plant resistance to environmental stress factors [5][6][7].
Previously we have researched biosynthesis of the phytohormones auxins and cytokinins by free-living bacteria Azotobacter chroococcum and symbiotic strains of Bradyrhizobium japonicum with various effectiveness rates [8,9].However, the ability of these microorganisms to production phytohormonal compounds that inhibit the of plant growth and development, which include abscisic acid (ABA) and gaseous hormone ethylene, and their role in symbiosis functioning of remain researched insufficiently.Such studies are essential for development of the microbiological preparations-legume inoculants technology with combined properties, because in the composition of these preparations should be the optimal ratio of living cultures of nitrogen-fixing bacteria and their metabolites for efficient formation and functioning of symbiosis.
In this connection, the aim of the work was to research the production of phytohormones that inhibit the plants growth and development -ABA and ethylene by various nitrogen-fixing symbiotic efficiency biotechnological strains of B. japonicum under the conditions in vitro.

Materials and Methods
The objects of research were the nodule soybean bacteria strains with different nitrogen fixation activity under symbiosis conditions: highly effective biotechnological strains of B. japonicum UCM В-6018, UCM В-6023, UCM В-6035 and UCM В-6036, which were high virulent, formed active nitrogenfixing apparatus on soybean roots under symbiosis conditions and increase in the crop yield and the protein content in soybean seeds (strains are from the collection of Department of general and soil microbiology, Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, deposited in the Ukrainian collection of microorganisms, UCM).These strains are industrial and they used as producents to bacterial preparations (inoculants) for crop plants in the factory for the production of biological products in Ukraine.
In the research, in order to compare the biosynthesis ability of symbiotic bacteria of soybean, were used nonindustrial rhizobia such as: B. japonicum 21110 strain with small efficiency, which had low virulence, formed nodules with low nitrogen-fixing activity on soybean roots and had little effect on the crop yield and its quality (strain received in previous years from the collection of the All-Russian Research Institute of Agricultural Microbiology RAAS, St. Petersburg, Pushkin); and ineffective strain of B. japonicum 604k (the control strain), which had high virulence but formed nodules without nitrogen-fixing activity on soybean roots (strain granted in previous years, the Institute of Agriculture Crimea, NAAS).
Characteristics of the studied B. japonicum strains under conditions of establishment and functioning of symbiosis with soybean plants, which included such indicators as: the average number and mass of nodules and their nitrogenase activity per plant, were obtained in previous years by the Department of general and soil microbiology, Zabolotny Institute of Microbiology and Virology, NAS of Ukraine (Table ).
The cultivation of bacteria was performed in flasks with volume of 750 ml rocking (220/min) at 26-28 C during 72-96 hours in the liquid nutrient medium of Iswaran with such composition (g/l): mannitol -10.0; east extract -2.0; calcium gluconate -1.5;K 2 HPO 4 -0.5;MgSO 4 •7Н 2 О -0.2; NaCl -0.1; FeCl 3 •6Н 2 О -0.01; рН 7.2.The cultures of B. japonicum in the exponential growth phase (92-96 h) grown on the Iswaran medium were used as inoculums.The amount of inoculums was 5% of the medium volume.For separating the biomass the bacteria culture broth was centrifuged for 20 min at 9000 rev/min and 4 C.Bacterial cells were washed with saline three times in order to clean from exopolymers residues, each time they were centrifuged under the same conditions.The supernatants were used for further studies for the purpose of extraction of ABA, and the residue of cells was suspended in distilled water and then dried at 103-105 C in an oven until the constant weight was reached.The amount of absolutely dry biomass (ADB) of microorganisms was determined gravimetrically.
The extracellular ABA was isolated from the supernatant of B. japonicum by extraction of phytohormones with ethyl acetate at pH 3.0 [11].The obtained extracts were evaporated in vacuum at 40-45 C.The dry residue was dissolved in 5 ml of ethanol and transferred to micro test tubes.Ethanol extracts from the supernatant of the studied soybean rhizobia were used for accumulative thin-layer chromatography.The previous purification and the concentration of ABA were performed on plates with silica gel, mark Silufol UV 254 (Chemapol, Czech Republic), in the mixture of solvents applied subsequently: chloroform; 12.5% aqueous ammonia; ethyl acetate: acetic acid (20:1).The purified in that way extracts were separated on plates with silicon oxide Merck, №5554, F 254 (Germany) in the mixture of solvents: chloroform: ethyl acetate: acetic acid (100:100:1) [12].The quantitative detection was performed with a scanning spectrodensitometer ("Sorbfil", Russia).The amount of extracellular ABA was calculated in g per 1 g ADB of the producer.The standard was the synthetic ABA by Sigma-Aldrich (Germany).
The amount of ethylene produced by symbiotic nitrogen-fixing soybean bacteria was measured by gas chromatography.The bacteria were cultivated in flasks of 25 ml on solid synthetic medium slant of Iswaran, in which L-methionine (Met) was added with concentration of 0.1 g/l [5].The flasks were tightly closed with rubber stoppers and special metal clamps in order to prevent escape of the synthesized ethylene.The further cultivation of B. japonicum strains was performed for 72 hours at 28 C.After the cultivating of bacteria the gas mixture from the flasks was analyzed with a gas chromatograph ("Chrome-5", Czech Republic) with a flame ionization detector (a column with -oxide propionitrile).Recalculation was performed on the calibration schedule built according to ethylene dilutions.
All experiments were performed in 6 iterations.The results obtained were processed statistically using computer program Excel from licensed Microsoft Office 2010.In the

Results and Discussion
The study results revealed that the ABA production by symbiotic soybean bacteria under the conditions in vitro did not correlate with nitrogenase activity of the strains in symbiosis.It is noted that the ability to form large amount of this phytohormoneplant growth inhibitor is a strain property of high virulent biotechnological rhizobia.Thus, significant amounts of ABA (56.5-72.0μg/g ADB) are capable to synthesize both the highly effective microsymbionts of soybean B. japonicum UCM B-6035 and UCM B-6036 and the ineffective strain of B. japonicum 604k (Fig. 1).The ABA synthesis was almost 20 times higher in the effective industrial strains of B. japonicum UCM B-6035 and UCM B-6036 than in the other active industrial strains of B. japonicum UCM B-6018 and UCM B-6023.
According to the literature, this fact may indicate that in various strains of symbiotic soybean bacteria synthesized ABA can both increase and reduce activity of nitrogenase enzyme, which is directly involved in converting air nitrogen into amine nitrogen.Therefore, processing of legumes with biological preparations-inoculants, which contain large amount of bacterial ABA specifically, can negatively affect the formation of nodules and reduce the efficiency of symbiotic nitrogen fixation [6].At the same time, the ability of B. japonicum to production ABA is an additional factor that increases potential activity of these strains in infection of plants.Also, it is known that ABA can reduce high concentrations of growth stimulating substances [13].
It is known that ABA, on the one hand, is a phytohormonal adaptogen, therefore, it enhances the plant resistance to environmental stress factors.On the other hand, it stimulates nitrogenase activity by inhibiting production of nitric oxide in nodules [14].At the same time, ABA is a chemical factor that determines the pathogenicity of microorganisms in infection of plants [2,15].This phytohormone inhibits the plant growth and development and accelerates defoliation and fruit ripening [16].
The role of ABA synthesis as the hormonal exometabolites for microorganisms is not studied enough yet.In the literature the data on the role of this compound in plant-microbe interactions are presented mainly.
In particular, the role of ABA in legumerhizobia symbiosis also is not fully clear.The depressing effect of the hormone in formation of nodules was shown [17,18] that was associated with the decrease in content of flavonoids (daidzein, genistein, kumestrol) in soybean roots.There are the data that ABA can interact with phytohormones cytokinins at process of root cortical cell division inhibiting it [19].
After inoculation with rhizobia the formation of nodules on a soybean plant slows down under the influence of exogenous ABA and, on the contrary, the number of nodules increases abruptly at reducing concentration of the hormone under action of the specific inhibitor (9-cis-epoxycarotenoid dioxygenase).The observation on the root hair deformation revealed that ABA blocked the stage between the root hair swelling and its curling.It is assumed that in this way ABA controls the number of nodules on the plants roots [17].However, it was shown that ABA was not directly involved in system self-regulation of nodules number and inhibited their formation only locally [20].
We have obtained the data on small amounts of gaseous phytohormone-inhibitor of the plant growth ethylene that were produced by symbiotic soybean bacteria B. japonicum (Fig. 2).As it is evident from the results obtained, the level of ethylene synthesis in diverse biotechnological strains varies widely.Thus, highly effective industrial rhizobia strains of B. japonicum UCM В-6023, UCM В-6018 and UCM В-6035 have low rates of synthesis of this phytohormone.Instead, the ineffective strain of B. japonicum 21110 differs significantly in amount of synthesized ethylene, which is 75.2 times higher than the lowest value of B. japonicum UCM В-6035.The direct correlation between level of synthesis of the hormone and nitrogenase activity of the strains in symbiosis was not observed.According to the literature, the relatively low pool of ethylene in plant is important to the symbiotic bacteria, because this hormone significantly inhibits formation of nodules on the plants roots [21,22].Probably, this explains the relatively low level of ethylene synthesis by all studied strains of B. japonicum.
Ethylene plays the dual role in formation of nitrogen-fixing nodules: it can suppress their formation or under certain concentrations it can stimulate rhizobium infection [23,24].It is known that inhibition of ethylene synthesis contributes to an increase in number of nodules formed on the roots of peas, alfalfa and other legumes [7].
In order to study mechanisms that rhizobia use to control nodulation the gene of synthesis of enzyme 1-aminocyclopropane-1carboxylic acid (ACA) deaminase, inhibiting the formation of ACA-ethylene synthesis precursor in higher plants, was isolated and characterized in Rhizobium leguminosarum bv.viciae 128C53K.Due to the action of ACA-deaminase, bacteria can reduce ethylene synthesis in plants [24].
There is an information that ethylene, as a component of signaling system, takes part in bacterial infection in legumes and it is a negative regulator of the Nod-factor signaling [23,25].Increase of endogenous ethylene level in cells of the host plant under NO 3 can lead to further restrictions of nodulation in addition to regulatory impact (autoregulation) on these processes regarding ethylene, synthesis of which is initiated by Nod-factor.The impact of nitrate on ethylene synthesis is associated with the activation of ACA oxidase [24].However, the mechanism of ethylene influence on formation of legume-rhizobia symbiosis is not fully understandable.It was shown that ethylene inhibited all primary host plant responses to rhizobia infection including output of Ca 2+ into the cytosol [22].It is assumed that ethylene can affect the activation of Nod-factor signaling pathway.It can act as a secondary messenger, regulating nodulation in response to the nitrogen status of the host plant, and as a negative regulator of rhizobia infection [21,25].
Production of ethylene by rhizobia, in particular B. japonicum, was studied by cultivating the bacteria on the medium with of L-methionine, which resulted in increased phytohormone synthesis under these conditions.In addition, if L-methionine is present in apoplast or root exudates, the association of Bradyrhizobium with the plant can increase level of endogenous ethylene in a plant and thereby inhibit the formation of nodules [5].
Therefore, the synthesis of ABA and ethylene by symbiotic rhizobia of soybean under the conditions in vitro is a specific property of researched biotechnological strains.This process does not depend on their nitrogenase activity in nodules and further functioning of symbiosis.The ability to synthesize the phytohormones, which show the depressing effect on the plant growth and development, can be an additional factor that increases virulence of strains of B. japo nicum and can influence the growth of their pathogenicity in infection of plants.We can assume that ABA and ethylene of bacterial origin take part in the formation of legume-rhizobia symbiosis, and their role is primarily to stimulate cell division of roots tissues, which start the formation of symbiotic relationship and the creation of the optimal number of nodules for a plant.Besides that, excessive concentration of these compounds in bacterial biological preparations obtained from cultivation of B. japonicum and used for legume seeds processing before sowing, can significantly reduce quality of the obtained preparation and, accordingly, reduce the effectiveness of functioning of legume-rhizobia systems.
In addition, the results obtained are of practical importance for development of new biotechnologies for creation of efficient microbial preparations and inoculants for plant cultivation and further forecasting of microorganisms activity in agricultural ecosystems.

Characteristics of nitrogen-fixing strains of B. japonicum in the formation and functioning of symbiosis with soybean plants B. japonicum strains Average number of nodules per plant, pcs. The weight of nodules per plant, mg Nitrogenase activity of nodules, μmol С 2 Н 4 per plant for 1 hour
[10]ta are accordance with the work[10].
Table and the Figures the average values and standard errors (M ± m) are presented.Values of P < 0.05 were considered to be significant.