Сельскохозяйственная биология, 2012, № 1, с. 98-104.

doi: 10.15389/agrobiology.2012.1.98eng

УДК 633.11:631.847.2:[581.132+631.524.84

EFFECT OF PRETREATMENT OF SPRING WHEAT SEEDS BY LECTIN AND LECTIN-BACTERIAL COMPOSITION ON THE PHYSIOLOGICAL INDEXES OF PLANT DEVELOPMENT AND PRODUCTIVITY

E.V. Kirichenko

The effect of exogenous lectin and its composition with nitrogen-fixing bacteria Azotobacter chroococcum T79 on the contents of chlorophyll and carbohydrates in the leaves of plants, formation of vegetative mass, grain productivity of wheat and nitrogenase activity of the rhizospheric microorganisms have been investigated. It was shown, that pretreatment of seeds by lectin and lectin-bacterial composition promoted the increase of chlorophyll and carbohydrates in the leaves, nitrogenase activity of the rhizospheric complex of microorganisms as well as accumulation of plants biomass and grain productivity of spring wheat.

Keywords: spring wheat, lectin, lectin-bacterial composition, chlorophyll, carbohydrates, rhizospheric microorganisms, nitrogenase activity, grain productivity.

Productivity is a complex feature of plants based on genetically determined processes of growth, morphogenesis, generative development and aging caused by many factors, the most important of which is photosynthetic activity depending on chlorophyll content and nitrogen supply (1). Phytohormones play a decisive role in regulation of plant growth and development (2). Exogenous action of bioactive substances, such as hormones, provide a significant activation of chlorophyll synthesis, photosynthetic processes in leaves and increase in productivity of agricultural crops (3, 4). An efficient way to improve yield capacity of plants is treating sown seeds with bacterial microorganisms as a source of biological nitrogen for plants and active producer of growth-promoting substances (hormones, vitamins, amino acids, etc.) (5).
Recent studies indicate that plant lectins and phytohormones are involved in regulation of growth and development of plants (6). Thus, treatment of wheat seedlings with wheat germ agglutinin (WGA) stimulates mitotic activity in meristematic cells of roots (7). The interaction of WGA and 24-epibrassinolide in regulation of cell division in wheat roots has been described (8). Previously, it has been shown (9-11) that pre-sowing treatment of soybean seeds with a soybean-specific lectin and its combination with Bradyrhizobium japonicum 634b activates plant growth, formation of phytomass and yield of seeds, along with the development and functional activity of nodule bacteria and rhizospheric diazotrophic microorganisms.
The purpose of this work was to study the effects of pre-sowing treatment of wheat sown grain with wheat lectin and its combination with a nitrogen-fixing bacteria Azotobacter chroococcum T79 on contents of chlorophyll and carbohydrates in leaves, the formation of a vegetative mass and grain yield, as well as the number and activity of nitrogen-fixing rhizospheric microorganisms.
Technique. Spring wheat (Triticum aestivum L.) cultivars Rannyaya 93 (high productive) and Kollektivnaya 3 (medium productive) were grown during 2003-2005 in vegetation vessels (Wagner type, volume 9 kg) on a substrate (soil: sand - 3:1) with Pryanishnikov nutrient medium (half rate of mineral nitrogen) (12) in natural light and temperature conditions of a vegetative test site (replication – 7-fold). Agrochemical characteristics of soil: light-gray loam, pHKCl = 6,9; contents of N, P and K - respectively, 1,32; 0,28 and 0,81%.
In 1 h before sowing the grains were soaked in wheat lectin solution (the commercial preparation of high purity WGA by “Lektinotest”, Lviv, Ukraine) with the concentration of 10 ug/ml, as well as in the composition containing WGA and a suspension of nitrogen-fixing bacteria A. chroococcum T79 (1:1, final content of lectin 5 ug/ml, the titer of bacteria 108 cells/ml) (experimental variants) and in water (control variant). The strain A. chroococcum T79 was isolated using analytical selection from chernozem soil of the Poltava Region of Ukraine (13). The bacterial culture was grown on Ashby agar medium and washed off with sterile water (5).
Samples of plants and rhizosphere soil were collected during a tillering and in a period of booting – early heading. A crude phytomass was determined by weighing, chlorophyll content (mg/g wet tissue of leaves) – as described by Arnon after extraction with dimethylsulfoxid (14), the content of carbohydrates (percentage calculated to glucose) – according to Kh.N. Pochinok (15). Total number of microorganisms in rhizospheric soil was established by serial dilutions (16), identity to groups of nitrogen-fixing microorganisms – by inoculation on nitrogen-free Ashby medium (5) with following account of colonies. Nitrogen-fixing activity of rhizospheric bacteria was measured by an acetylene method of R.W.F. Hardy et al. (17) on the device Chromatograf 504 (Poland). Yield structure and productivity were estimated in the phase of full ripeness.
Statistical analysis of data was performed in the program Statgraphics Plus v. 3.0, arithmetic means and standard errors calculated for at least three biological replicates are shown in tables.
Results. Pre-sowing treatment of seeds with lectin and lectin-bacterial composition provided a positive effect on chlorophyll accumulation in plants of spring wheat. Thus, in the variant of only lectin, during the first half of a growing season (from tillering and booting to early heading), chlorophyll content in leaves of cv Rannyaya 93 exceeded the values of control plants by, respectively, 6 and 18% , in cv Kollektivnaya 3 – by 4 and 20%. In the variant of lectin-bacterial composition, a similar increase was observed: in cv Rannyaya 93 – resp., by 8 and 28%, in cv Kollektivnaya 3 - by 9 and 18% (Table 1).

The action of lectin and lectin-bacterial composition was compared in respect to the chlorophyll content in leaves in different vegetation phases and no significant differences were found. Although a half content of lectin in lectin-bacterial composition compared with a pure preparation (respectively, 5 and 10 ug/ml), the effect wasn’t much different or even was somewhat higher than in pure WGA, probably owing to the bacterial component. It was shown that co-incubation of nitrogen-fixing bacteria with lectin provides the activation of metabolic processes in pure culture bacterial cells, which results in increased synthesis of proteins, enzymes and products of nitrogen metabolism (18), the growth hormone indole-3-acetic acid (19), extracellular polysaccharides (20); in symbiosis with legumes, there occurs a stimulation of nodulation capacity of bacteria, nitrogen-fixing activity of root nodules and efficacy of symbiotic systems (11, 21, 22).

Increased chlorophyll content contributes to activation of photosynthesis, the formation of plastic substances and ve-vegetative mass of plants (1). It was found that both lectin and lectin-bacterial composition provided accumulation of carbohydrates in leaves (Table 2). During a tillering, cv Rannyaya 93 manifested the increase in this characteristic by, respectively,  1,2 and 1,4 times, cv Kollektivnaya 3 – by 3,4- and 3,7-fold, which indicates that different wheat cultivars develop an unequal reaction to seed treatment with lectin. Plants in both experimental variants exceeded control in formation of green phytomass (Table 1). The variants of pure lectin and lectin-bacterial composition differed in this characteristic probably owing to the additional influence of bacterial component, since a similar positive effect has been observed at pre-sowing treatment of wheat seeds with A. chroococcum T79. Previously, a pre-sowing seed inoculation with this strain has provided 10% increase in grain yield of cv Rannyaya 93 during a field experiment (23). The efficiency of lectin composition with A. chroococcum T79 vs. pure bacterial strain were compared and the first was found to be superior as it caused a long-term effect manifested throughout a growing period (stimulation of rhizogenesis, formation of aboveground phytomass and grain yield, as well as growth of population of rhizospheric nitrogen-fixing microorganisms) (24) .
Thus, wheat lectin and lectin-bacterial composition provide an intense formation of vegetative mass due to increase in content of green photosynthetic pigments in leaves and carbohydrate synthesis. The accumulation of carbohydrates since earliest stages of plant development may also be associated with increased activity of enzymes related to photosynthetic carbon assimilation. It is known (25) that in vitro a purified preparation of PLCI (pigment-lectin complex of photosystem I) of triticale caused a significant (40-50%) activation of ribulosobiphosphate carboxylase (RBPCO - the key enzyme of dark phase of photosynthesis) isolated from the stromal fraction of chloroplasts from leaves of triticale and poplar, which changes were detected in the light as well as in darkness. The replacement of a purified PLCI by PLCI-enriched fraction of stromal thylakoids decreased enzyme activity compared with pure PLCI, which effect was light-dependant. Lectin stimulation of enzyme activity of PBPCO has been proved upon the hapten of lectin isolated from the stroma of triticale thylakoids – galacturonic acid: when the pre-incubation of a purified PLCI with the hapten, no effect of PLCI in respect to PBCO was observed (25).

 
Photosynthetic activity of plants is known to be essentially affected by rhizosphere microorganisms fixing molecular nitrogen and improving nitrogen nutrition of plants, as well as exogenous bioactive substances (3-5). The activity of associative nitrogen fixation in this case increases by 2-5 times, which can be the result of increased accumulation of chlorophyll (5).
Studying correlations of a chlorophyll content in leaves with bacterial nitrogen-fixing activity and number of rhizospheric diazotrophs, it was found (Table 3) that seed treatment with lectin provided the increase in bacterial nitrogenase activity in 1,7 and 1,4 times while in chlorophyll content - by 14 and 16% for cv Kollektivnaya 3 and Rannyaya 93 respectively. At the same time, the number of rhizospheric nitrogen-fixing microorganisms increased in, respectively, 1,6 and 1,2 times (Table 3). Lectin-bacterial composition caused in both cultivars an outstanding growth in number of nitrogen-fixing bacteria – in 2,4 and 1,6 times relative to control in cv Rannyaya 93 and Kollektivnaya 3, respectively, while the rise in bacterial nitrogenase activity in 1,2 and 2,0 times and in chlorophyll content - by 16 and 18% (Table 3).
The increase in nitrogen-fixing ability of rhizospheric diazotrophs can be associated with lectin stimulation of their physiological activity, which is suggested by works when increased bacterial growth was obtained in both pure culture and in the rhizosphere (10, 20, 26), as well as rise in activity of bacterial nitrogenase (10, 18, 27). In lectin-bacterial composition, lectin provides a direct action on A. chroococcum T79 resulting in an increased number of bacterial cells (26). The effect of composition on rhizospheric microorganisms can be manifested during the introduction of lectin-activated bacteria strain T79 into plants’ rhizosphere and a following direct action on rhizospheric microorganisms caused by lectin not associated with the bacterial strain (28).
As a result, rhizospheric nitrogen-fixing bacteria developed increased N2-fixation, which improved nitrogen supply of plants with biologically available nitrogen. This intensifies their photosynthetic activity providing more photoassimilates (carbohydrates, organic acids, etc.) a part of which along with root metabolites enters soil and rhizosphere, which, in turn, affects the development and physiological activity of rhizospheral microbiota.

 
The other (major) part of photoassimilates is involved in formation of vegetative biomass and productive potential of plants. Thus, pre-sowing seed treatment with lectin and the lectin-bacterial composition provided increased grain yield in the studied cultivars (Table 4). The analysis of yield structure reveals the increase in ear length and weight, number and weight of grains per ear, as well as harvest index (the ratio of grain weight to vegetative weight of a plant). Unequal effects of lectin and lectin-bacterial composition on productivity of different wheat varieties can be explained by their genetic features in terms of variety responsiveness to specific preparations, secretory capacity of germinating seeds, qualitative and quantitative composition of exometabolites seed and root, the degree of binding of lectin and bacteria with a surface of seeds and their effects on the development of seeds and rhizospheric microbiota. Nevertheless, both lectin and lectin-bacterial composition were found to provide a prolonged effect of increased grain productivity in wheat.
Thus, pre-sowing treatment of wheat grains with wheat lectin as well as the composition of wheat lectin and nitrogen-fixing microorganisms Azotobacter chroococcum T79 has resulted in increased contents of chlorophyll and carbohydrates in leaves, formation of the vegetative mass and grain productivity in wheat, stimulated the development of agronomically-useful soil microorganisms and their nitrogen-fixing activity in rhizosphere of plants. In the case of lectin-bacterial composition, a bacterial component allows to decrease the content of wheat lectin while maintaining the efficiency, which reduces the cost of seed pre-treatment (composition) compared with pure lectin.

REFERENCES

1. Gulyaev B.I., Photosynthesis and Productivity of Plants: Problems, Achievements and Prospects for Research, Fiziologiyaibiokhimiyakul’turnykhrastenii, 1996, vol. 28, no. 1-2, pp. 15-35.
2. Derfling K., Gormonyrastenii. Sistemnyipodkhod(Plant Hormones. A Systems Approach), Moscow, 1985.
3. Chernyad’yev I.I., Photosynthesis and Cytokinins, Prikladnayabiokhimiyaimikrobiologiya, 1993, vol. 29, no. 5, pp. 644-673.
4. Kartashov I.M., Kholodenko N.Ya. and Muzafarov E.N., Effects of Kinetin and Abscisic Acid on the Activity of Adenylate Kinase in Pea Chloroplasts, Prikladnayabiokhimiyaimikrobiologiya, 1999, vol. 35, no. 4, pp. 463-467.
5. Patika V.P., Kots’ S.Ya., Volkogon V.V. et al., Biologichniiazot(Biological Nitrogen), Kyiv, 2003.
6. Rudiger H. and  Gabius H.-J., Plant Lectins: Occurrence, Biochemistry, Functions and Applications, Glycoconj. J., 2001, vol. 18, pp. 589-613.
7. Bezrukova M.V., Kil’dibekova A.R., Aval’baev A.M. and Sakirova F.M., Wheat Germ Agglutinin Participates in Regulation of Cell Division in Apical Root Meristem of Wheat Seedlings, Tsitologiya, 2004, vol. 46, no. 1, pp. 35-38.
8. Bezrukova M.V., Aval’baev A.M., Kil’dibekova A.R., Fatkhutdinova R.A. and Shakirova F.M., Interaction of Wheat Lectin with 24-Epibrassinolide in the Regulation of Cell Division in Wheat Roots,  Dokl. RAN, 2002, vol. 387, pp. 276-378.
9. Kirichenko E.V., Titova L.V., Kots’ S.Ya., Zhemoyda A.V., Omel’chuk S.V. and Mar’yushkin V.F., The Effect of Soybean Lectinon the Yielding Capacity of Soybeans, Agrokhimiya, 2004, no. 11, pp. 58-62.
10. Kirichenko E.V. and Titova L.V., The Influence of Exogenic Soybean Lectin on the Development and Nitrogen-Fixing Activity of the Root Nodules and Diazotrophic Microorganisms in a Rhizospheric Zone of Plants, Fiziologiyaibiokhimiyakul’turnykhrastenii, 2005, vol. 37, no. 2, pp. 139-146.
11. Kirichenko E.V. and Titova L.V., Soybean Lectin as a Component of a Composite Biopreparation Involving Bradyrhizobium japonicum 634b, Prikladnaya biokhimiya i mikrobiologiya, 2006, vol. 42, no. 2, pp. 219-223.
12. Grodzinsky A.M. and Grodsinsky D.M., Kratkiispravochnikpofiziologiirastenii(A Quick Reference Guide on Plant Physiology), Kyiv,  1971.
13. Kots’ S.Ya., Titova L.V., Kirichenko O.V., Omel’chuk S.V. and Zhemoyda A.V., ShtambakteriiAzotobakterchroococcumT79 dlyaoderzhannyabakterial’nogodobrivapidsoyu(The Strain AzotobacterchroococcumT79 for Obtaining a Bacterial Fertilizer for Soybean), Patent of Ukraine  ¹ 62820А C05F11/08, C12N1/20, Publ. 15.12.2003, Bull. ¹ 12.
14. Hiscox J.D., Israelstam R.J., The Method for the Extraction of Chlorophyll from Leaf Tissue without Maceration, Can. J. Bot., 1979, vol. 57, no. 12, pp. 1332-1334.
15. Pochinok Kh.N., Metodybiokhimicheskogoanalizarastenii(Methods for Biochemical Analysis of Plants), Kyiv, 1976.
16. Praktikum po mikrobiologii: Uch. pos. (Practice on Microbiology: Student’s Manual), Netrusov A.I., Ed., Moscow, 2005.
17. Hardy R.W.F., Holsten R.D., Jackson E.K. and Burns R.C., The Acetylene-Ethylene Assay for N2-Fixation: Laboratory and Field Evaluation, PlantPhysiol., 1968, vol. 43, no. 8, pp. 1185-1207.
18. Antonuk L.P. and Ignatov V.V., About the Role of Agglutinin from Wheat Germs in Plant-Bacteria Interaction: Hypothesis and Experimental Data for Its Support, Fiziologiya rastenii,2001, vol. 48, no. 3, pp. 427-433.
19. Iosipenko A.D., Sergeeva E.I., Antonuk L.P. and Ignatov V.V., Effects of Wheat Lectin on the Synthesis of Indolyl-3-Acetic Acid in Azospirillum brasilensе sp. 245, Dokl. RAN, 1994, vol. 336, no. 4, pp. 554-561.
20. Kosenko L.V. and Mandrovskaya N.M., Effect of Pea Lectinon the Growth of Pea Microsymbionts and Exoglycan Biosynthesis, Mikrobiologiya, 1998, vol. 67, no. 5, pp. 626-631.
21. Kirichenko E.V., Biological Activity of Legume Lectins in Respect to Nodule Bacteria in Mat. konf. “Fundamental’nye i prikladnye aspekty issledovaniya simbioticheskikh system” (Papers of Sci. Congress “Fundamental and Applied Aspects of Symbiotic Systems”), Saratov, 2007, p. 88.
22. Lodeiro A.R., Lopez-Garcia S.L., Vazquer T.E.E. and Favelukes G., Stimulation of Adhesiveness, Infectivity and Competitiveness for Nodulation of Bradyrhizobium japonicum by Its Pretreatment with Soybean Seed Lectin, FEMS Microbiol. Lett., 2000, vol. 188, no. 2, pp. 177-184.
23. Kots’ S.Ya., Titova L.V., Kirichenko O.V., Omel’chuk S.V. and Zhemoyda A.V. , Efektivnist’ preparativrizosfernikhdiazotrofivpriviroschuvanniyaroї pshenitsi(Efficacy of Biopreparations of Rhizospheric Diazotrophic Bacteria on Spring Wheat) in Zhivlennyaroslin: teoriyaipraktika, Kyiv, 2005, pp. 306-314.
24. Kirichenko E.V., Zhemoyda A.V. and Kots’ S.Ya., Effectof a Plant-Bacterial Composition on the Yielding Capacity of Spring Wheat, Agrokhimiya, 2005, no. 10, pp. 41-47.
25. Aleksidze G.Ya., Litvinov A.I. and Vyskrebentseva Je.I., The Model of Calvin Cycle Enzyme Organization on Thylakoid Membranes with the Involvement of the Photosystem I Lectin, Fiziologiya rastenii, 2002, vol. 49, no. 1, pp. 155-159.
26. Karpati E., Kiss P., Ponyi T., Fendrik I., De Zamaroczy M. and Orosz L., Interaction of Azospirillum lipoferum with Wheat Germ Agglutinin Stimulates Nitrogen Fixation, J. Bacteriol., 1999, vol. 181, pp. 3949-3955.
27. Kirichenko E.V. and Titova L.V., Effects of Plant Lectins on Growth of Cultured Soil Microorganisms, Agroekologichnii zhurnal, 2005, no. 4, pp. 52-56.
28. Kirichenko O.V., Zmina aktivnosti lektinu pshenitsi ta supenu iogo vzajemodії a riznimi komponentami pri stvorenni kompozitsii lektinovoї prirodi (Changes in Activity of Wheat Lectin and Its Interaction with Different Components during a Creation of a Composition of Lectin Nature), Ukra¿ns’kii biokhimichnii zhurnal, 2006, vol. 78, no. 6, pp. 105-112.