Biological activity of wheat germ agglutinin relative to spring wheat plants under the influence of hapten N-acetyl-D-glucosamine

© 2020 O. V. Kyrychenko; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 581.192


Introduction
The use of ecological biotechnology elements (microbial inoculants and biologically active substances of natural origin) in the rural economy in order to increase the crop productivity of culture plants is the urgent issue of sustainable agriculture. Microbial biotechnology and biologically active substances of natural origin (plants and microbial metabolites) are employed in agriculture to fulfill environmental and industrial tasks [1,2]. Phytolectins are natural substances, the products of plants metabolism [3,4]. Lectins are the molecules with a wide range of biological activities [5][6][7][8][9]. They are glycoproteins or oligomeric proteins with one or more sugar-binding sites per subunit. Lectins bind reversibly with specific sugars [10]. Carbohydrate specificity is a fundamental characteristic of the phytohemagglutinin molecules [10,11]. Wheat germ agglutinin (WGA) belongs to the group of N-acetyl-Dglucosamine (GlcNAc)-binding plant lectins. WGA has two primary and two secondary independent sugar-binding sites and possesses sugar binding specificity for two types of N-acetylated sugars -N-acetyl-D-glucosamine and N-acetylneuraminic acid [4].
Recently, it has been shown the inducing effects of WGA on the RNA amount, enzyme (peroxydase and catalase) activity of antioxidant system of plant protection, endogenous lectin activity and flavonoids content in wheat leaves and the content of plant hormones (cytokinins and IAA) as well as on the nitrogen fixation activity of rhizospheric microorganisms [7,12].
The aim of the present investigation was to study the biological activity of wheat germ agglutinin (WGA). It was investigated an influence of N-acetyl-D-glucosamine (hapten of WGA, GlcNAc) at the pre-sowing treatment of spring wheat seeds on the wheat plants characteristics: growth parameters, chlorophyll (Chl) a+b content and grain productivity of Triticuma estivum L. in greenhouse conditions.

Material and Methods
Spring wheat (Triticuma estivum L.) cv Rannya 93 plants [13] were grown on sand soil with components of Pryanishnikov nutrient medium containing (g/kg): 0. 12  Preparations of wheat germ agglutinin (100 nM) and aminosaccharide N-acetyl-Dglucosamine (100 mM) ("Lectinotest", Lviv, Ukraine) were used [14]. The ratio for composition of lectin with hapten was 1:1, where lectin was pretreated with hapten for 1 h. Water was used as control, while WGA and WGA+GlcNAc -as experimental variants for pre-sowing treatment of wheat seeds for 1 h.
The shoot weight (fresh weight, FW) and chlorophyll (Chla+b) content in wheat leaves were estimated at seedling development (10 day-old plants), tillering (30 day-old plants), boot development (45-50 day-old plants) and head emergence -flowering (55-60 day-old plants) of wheat vegetation phase [15]. Chl(a+b) content in wheat flag leaves was determined according to Arnon after extraction of leaves with dimethylsulfoxide [12]. The level of Chl was measured in four replications and expressed in mg g -1 FW leaves. The grain productivity (dry weight, DW of plants and grains) of spring wheat was estimated at ripening phase (90-100 day-old plants) of plants vegetation [15]. Yield structure parameters (weight of one spike, grain number and weight per spike, weight of 1000 grains and harvest index) were estimated.
Statistical evaluation of the results was performed according to Statgraphics software statistical package 5.0. All data presented in the paper are Mean ± standard error of mean (SEM).

Results
The obtained results demonstrate that presowing treatment of spring wheat seeds with WGA has stimulated the growth processes and formation of plants biomass, Chl (a+b) content in wheat leaves and wheat yield, but GlcNAc has reduced the stimulatory effect of lectin at all developmental phases under greenhouse conditions (Table 1-3). Shoot mass formation (Table 1) increased by 10-24 %, while GlcNAc has inhibited a positive biological effect of WGA at different phases of wheat plants ontogenesis by 5, 19, 12 and 6 %. Hapten had a significant reducing effect on the wheat lectin biological activity regarding growth of spring wheat plants in the phases of tillering and boot development . At the same time no difference was observed in the phases of seedling develop ment.
Pre-sowing treatment of spring wheat seeds with WGA has increased Chl (a+b) content in the wheat leaves at the phases of seedling development by 21 %, of tillering by 38 %, of boot development phase by 26 %, of head emergence -flowering by 19 % (Table 2). Hapten had a significant inhibition effect on wheat lectin at the tillering and boot development phases of wheat plants: caused a reducer in Chl content by 18 and 11 % compared with effect of WGA, however, no difference was observed in the wheat at the seedling development and the head emergence -flowering phases.
The analysis of the yield structure has demonstrated that the grain productivity of spring wheat was enhanced due to an increase in the weight of spikes by 25 %, number and weight  (Table 3). An increase in the DW of 1000 grains was insignificant (by 6 %). The harvest index increased by 14 %. This effect suggests that the potential maximum of wheat plants formed in the presence of WGA was directed rather towards the production of wheat grains but not to the plant vegetative mass. GlcNAc has inhibited the activation of biological effects of WGA regarding to spring wheat productivity by 12 % (weight of one spike), by 8 % and 13 % (number and weight of grains per spike respectively). However, no difference has been shown in the values of 1000 grains and harvest index in its variants (by 4 % and 3 % respectively).
The intensification of plant shoot formation (Table 1) and chlorophyll content in leaves ( Table 2) as well as the spring wheat yield at WGA use for pre-sowing treatment of seeds (Table 3) was shown in greenhouse experiments during three years.

Discussion
Our recent results provide an evidence of the considerable role of exogenous phytolectins as regulators of plant growth and development and physiological activity of the soil nitrogenfixing microorganisms [2,7,12]. The response of spring wheat and soil rhizospheric nitrogenfixing microorganisms to the pre-sowing treatment of seeds by wheat germ agglutinin was expressed in the metabolic changes of plants: an increase in RNA amount and endogenous lectin activity in wheat seedlings and leaves, enzyme activity of antioxidant system of plant protection (peroxydase and catalase), endogenous lectin activity and flavonoids content and the amount of endogenous plant hormones cytokinine and auxine nature in wheat leaves [7,12], enhanced accumulation of plant bio-   (Table 1) and an increase in Chl (a+b) content in the wheat leaves (Table 2) as well as the nitrogen-fixing capacity of the rhizospheric microorganisms [2,7]. As a result, the wheat grain productivity increased ( Table 3).
Hapten of WGA, aminosaccharide GlcNAc, binds to the active centers of wheat lectin on the level of molecular interaction [14] and partially decreases the activation effects of WGA on the plants physiological and biochemical parameters. It is one of the arguments in favour of the lectin nature of these effects. It is know that lectins are the molecules exhibiting a wide range of biological activities regarding micro-(virus, bacterium, fungi) and macroorganisms (insects, plants, animals) [2,5,7,8,[16][17][18][19]. Both endogenous and exogenous lectins have many physiological functions in the plants [3, 4, 6-8, 16, 18]. Literature data confirms the results of our investigation, which have shown that pretreatment of WGA with GlcNAc has inhibited the positive effect of lectin on the wheat plants. It was shown that haptens of lectins blocked the lectin effects on the metabolism activity, physiological and biochemical parameters (like enzyme activities, membrane potential e. a.) of plants. It was reported that treatment of Nicotiana tabacum pollen grains with lectin concanavalin A (Con A) applied at concentrations of 10-1000 µg ml -1 has induced hyper polarization of plasma membrane in the vegetative cells and has enhanced pollen grain germination. Con A at a concentration of 100 µg ml -1 increased the intracellular pH by 0.3 units. These effects of Con A were blocked with saccharide methylmannopyra no side at a concentration of 100 mM [16]. Pretreatment of the bacterial lectins with L-fucose has decreased the effects of lectins [17]. It was shown that incubation of bacterial lectins isolated from the microsymbiont of wheat plant -soil nitrogen-fixing microorganisms Azospirillum brasilense sp.7 and its mutant defective in lectin activity (A. brasilense sp.7.2.3) with the exocomponent, membrane and apoplast fractions of wheat root increased the enzyme activities of α-glucosidase, β-glucosidase and β-galacto sidase. Lectins of wildtype and mutant strains had stronger stimulatory effect on the activities of all exocomponent fraction enzymes studied and of the apoplast fraction of β-glucosidase.
Thus, our data (table 1-3) have demonstrated that pre-treatment of WGA with N-acetyl-D-glucosamine changes the biological activity of protein at the pre-sowing treatment of wheat seeds in relation to the spring wheat plants. It is in agreement with previously demonstrated literature data on the inhibitory effect of haptens on the biological activity of lectins. Our research completes and provides the additional evidences for the lectin nature of stimulation effect of the molecules as the biologically active ones on the growth, development and grain productivity of spring wheat plants.

Conclusions
The biological activity of WGA at the presowing treatment of spring wheat seeds was expressed in the intensification of plant growth and development, the active accumulation of chlorophyll in leaves and an increase in the wheat grain productivity. N-acetyl-Dglucosamine -hapten of wheat lectin had a significant reducing effect on the wheat lectin biological activity that resulted in inhibition of positive lectin effects on physiological and Біологічна активність аглютиніну зародків пшениці щодо рослин пшениці ярої за дії гаптену N-aцетил-D-глюкозаміну