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Journal of Stress Physiology & Biochemistry, Vol. 20, No. 2, 2024, pp. 99-106 ISSN 1997-0838 Original Text Copyright © 2024 by Muslumova, Azizov and Farajov
ORIGINAL ARTICLE
OPEN/J) ACCESS
Humic Substances Antistress Activity at the Corn
Seedlings
Z. H. Muslumova 1*, I. V. Azizov 2, M. F. Farajov 1
11nstitute of Radiation Problems, Ministry of Science and Education, Republic of Azerbaijan
2 Institute of Molecular Biology and Biotechnologies, Ministry of Science and Education, Republic of Azerbaijan
*E-Mail: zohre.biologist@gmail.com
Received February 14, 2024
Under model conditions, the effect of potassium humate on some indicators in corn seedlings obtained from gamma-irradiated seeds was determined. A physicochemical analysis of potassium humate obtained from pine wood waste was carried out. In field and laboratory experiments, we studied the effect of potassium humate solutions on the morphological and biochemical parameters of corn seedlings obtained from irradiated seeds at doses of 50 and 100 Gy. In all experiments, solutions of potassium humate had a positive effect on the dynamics of growth and development of corn plants grown from irradiated seeds. The effect of potassium humate solutions on chlorophyll and carotenoid pigments, and on the photosynthetic activity (maximum quantum yield of PSII) of plants was studied. It has been established that the treatment of seeds with a 0.1% solution of potassium humate before irradiation reduces the amount of malondialdehyde, a product of lipid peroxidation. Seed treatment with 0.1% and 0.01% solutions of potassium humate before irradiation caused a significant reduction in the harmful effects of ionizing radiation on seedlings of corn plants.
Key words: Zea mays, potassium humate, chlorophyll pigments, irradiated seeds, malonic di-aldehyde, radioprotective activity, lipid peroxidation
Humic substances are widely used in agriculture as organic fertilizers. Various salts of humic acids are also used as growth and development stimulants. The stimulating effect of humic compounds on the growth and development of plants, increasing their resistance to adverse environmental factors has been sufficiently studied (Van Stempvoort et al., 2005; Holman, 2002). Soluble forms of humates in small concentrations significantly stimulate the growth and development of plants, increase the supply of nutrients to plants, activate protein and carbohydrate metabolism, and increase crop yields (Stevenson, 1982). We studied the effects of potassium humate on growth and development, on the morphological and physiological parameters of maize (Zea mays) variety "Zagatala 68" under conditions of radiation stress. It is known that ionizing radiation ultimately causes a violation of the integrity of cellular structures. The radioprotective properties of potassium humate were studied under model field conditions. There are a lot of scientific works on the use of humic substances in the rehabilitation of contaminated soils (Muslumova, 2015). The use of humic acids with other organic additives has a positive effect on the yield and quality of corn, as well as on soil properties and the availability of elements necessary for plant growth and development (Essam, 2020; Muslumova, 2013). In studies using various concentrations of humate solutions, the highest values of all studied indicators of corn plants were recorded -plant height, shoot diameter (cm), weight of 100 grains (g) and yield index (HI%) (Awwad, 2015). Thus, obtaining and studying the biological activity and radioprotective properties of humic substances is a very relevant direction.
MATERIALS AND METHODS
To obtain humate K, the feedstock - peat - was treated with 3% KOH solution with constant stirring at temperature of 35-40 degrees for 5 hours. After that, the dissolved sodium and potassium humates was separated in a centrifuge. The objects of research were maize seeds of the "Zagatala 68" variety. To study the radioprotective properties of humates, maize seeds
were treated with solutions of sodium and potassium humate for 15 hours. Then the seeds were irradiated with doses of 50 and 100 Gy using the URi (K-25) device. In experiments to study the effect of sodium and potassium humate on the dynamics of growth and development of seedlings were used 0,1%, 0,01% and 0,001% solutions. Plants were grown in the saline environment.
These experiments included a study of the growth dynamics of maize (Zea mays) seedlings obtained from irradiated corn seeds at doses of 50 and 100 Gy. Potassium humate was obtained from pine needle compost. We studied the percentage of humus in the compost and the elemental composition of the potassium humate. Before irradiation, maize seeds were treated with humate solutions at the concentration of 0.1% and 0.01%. The amount of chlorophyll a (662 nm), chlorophyll b (644 nm), carotenoids (440 nm) pigments and malondialdehyde (532 nm) determined spectrophotometrically (Multiscan Go, Germany). The maximum fluorescence quantum yield of maize seedlings was measured on a MINI-PAM fluorometer (Germany). Statistical processing of the results was carried out using Microsoft Office Excel and the Statistica software package. Grade the reliability of the obtained results was carried out on the basis of the calculation of the Student's criterion.
RESULTS AND ITS DISCUSSION
Pine needles contain valuable biological components, phytohormones, vitamin C, tannins, anthocyanins, alkaloids, terpenes, macro and microelements - calcium, iron, nickel, cobalt, phosphorus, manganese and zinc. Pine needle essential oil showed remarkable antioxidant activity in scavenging free radicals, in lipid peroxidation, and in reducing power assays. The essential oil showed strong antimicrobial activity with minimal inhibitory concentrations and minimal bactericidal concentrations (Qualls et al., 2003; Xavier de Campos et al., 2014) To obtain potassium humate, we used pine needle compost as a raw material. Pine waste composts are readily available raw materials. They can be harvested from the soil of pine forests. Composts can also be prepared in reactors. This can become a promising technology for the
utilization of organic household and wood waste (Zeng et al., 2012; Zeng et al., 2011). We conducted the physico-chemical analysis of the compost to determine the organic and inorganic composition.
At the beginning of the experiments, we studied the effect of potassium humate solutions on the germination of seeds irradiated at doses of 100 Gy. The germination of seeds not treated with humate solutions decreased to 55-65%. The germination of seeds irradiated at a dose of 100 Gy was 81% on the variant treated with 0.01% humate solutions, 84% on the variant treated with 0.1% potassium humate solutions. We studied the effect of potassium humate solutions on the amount of malonic dialdehyde, an indicator of lipid peroxidation, on chlorophyll pigments and carotenoids, and on the fluorescent indices of leaves (photosystem II) in seedlings.
The results obtained from the experiments performed indicate that the use of potassium humate solutions prevents damage to corn seedlings grown from irradiated seeds, compared with seedlings obtained from irradiated samples not treated with potassium humate solutions. Irradiation of corn seeds at dose of 100 Gy markedly inhibited the development of seedlings. A positive effect was observed in experiments to study the effect of humate solutions on the dynamics of growth and development of plants. Seedlings obtained from seeds treated with a 0.01% solution of potassium humate (PH) showed the best results in all stages of development of growth and development throughout the entire development stage.
As can be seen from Figure 1, growth and development of seedlings obtained from seeds treated before irradiation at a dose of 50 Gy with a 0,1% and 0.01% solutions of the PH increased by 13-15%, especially in the final stages compared to the irradiated variant.
Irradiation of corn seeds at a dose of 100 Gy noticeably inhibits the growth and development of plants (Figure 2) When seeds were treated with 0.1% and 0.01% solutions of the potassium humate (PH) before irradiation at a dose of 100 Gy, a noticeable effect of the complex on the dynamics of plant development was observed. In these experiments, we observed that the
growth and development of plants obtained from the seed complex treated with 0,1% and 0.01% solution increased by 15-18% at all stages compared to the irradiated variant.
It is known that radiation stress also affects the biosynthesis of photosynthetic pigments in plants. Many studies showed that chlorophyll pigments in irradiated plants were lower than in control plants. Photosynthetic pigments can be destroyed by gamma rays with a concomitant loss of photosynthetic capacity. In some experiments it was found that gamma irradiation leads to a greater destruction of chlorophyll b than chlorophyll a, due to a violation of its biosynthesis (Ling et al., 2008; Kiong 2008; Strid et al., 1990)
We investigated the effect of the complex on the content of photosynthetic pigments in maize seedlings. The study of the effect of the complexes on photosynthetic pigments was carried out in the spring stage of the experiment in seedlings obtained from seeds irradiated at doses of 100 Gy.
As can be seen from Figure 3, when corn seeds are irradiated with gamma irradiation at a dose of 50 Gy, the amount of photosynthetic pigments chlorophylls a and b decreases by 8-10%. In the irradiated variant of seeds at a dose of 50 Gy, both concentrations of the complex solution equally positively affect the amount of chl a and b and cause an increase in their biosynthesis by 4-6% compared to the irradiated variant. Irradiation at a dose of 100 Gy reduces the amount of chlorophyll a and b by 40% and 50%. In the variant, when the seeds were treated with 0.1% and 0.01% solutions of potassium humate, the amount of chlorophyll an increases by 3035%, chlorophyll b by 8-12% compared to the irradiated variant. In the variant with seed treatment with 0.01% potassium humate solution, the amount of carotenoids was also higher compared to the irradiated control in both seed irradiation doses. It can be said that the treatment of seeds of corn variety "Zagatala 68" with solutions of potassium humate before irradiation leads to the normalization of the biosynthesis of photosynthetic pigments and carotenoids.
We also studied the effect of potassium humate solutions on lipid peroxidation. Experiments to determine the effect of potassium humate solutions on lipid
peroxidation were carried out at the beginning of the development of corn seedlings in the first weeks. Gamma irradiation induce oxidative stress with overproduction of reactive oxygen species such as superoxide radicals, hydroxyl radicals, and hydrogen peroxides, which react rapidly with almost all structural and functional organic molecules including proteins, lipids, and nucleic acids causing disturbance of cellular metabolism. Reactive oxygen species react with almost all components of the cell. As a result, such an interaction contributes to free radical chain reactions. This ultimately leads to membrane lipid peroxidation (Montiller et al., 2004; Al-Rumaih et al., 2008). The free radicals that appeared after irradiation interact with the lipids of cell membranes. It is known that the process of lipid peroxidation is one of the important indicators of the effect of radiation on living organisms. When free radicals, subsequently formed under the action of radiation, interact with lipids of cell membranes, lipid peroxidation occurs. Lipid peroxidation causes the formation of several end products, one of which is malondialdehyde. The amount of formation of this product determined the degree of damage to the cells. Experiments to determine the effect of potassium humate solutions on lipid peroxidation were carried out at the beginning of the development of corn seedlings in the first weeks. Gamma irradiation induce oxidative stress with overproduction of reactive oxygen species such as superoxide radicals, hydroxyl radicals, and hydrogen peroxides, which react rapidly with almost all structural and functional organic molecules including proteins, lipids, and nucleic acids causing disturbance of cellular metabolism. Reactive oxygen species react with
almost all components of the cell. As a result, such an interaction contributes to free radical chain reactions. This ultimately leads to membrane lipid peroxidation (Moghaddam et al., 2020; Murchie et al., 2013)
Figure 4 shows that irradiation at a dose of 100 Gy has a positive effect on the yield of malonic dialdehyde, a product of lipid peroxidation. Seed treatment before irradiation with a 0.1% solution of potassium humate reduces the yield of malondialdehyde by 25-30% in the first weeks. Based on the results obtained, it can be concluded that potassium humate solutions lead to a decrease in the level of the product of lipid peroxidation -malondialdehyde and the maintenance of a normal concentration of photosynthetic pigments.
In the next experiment, we measured the chlorophyll fluorescence in corn leaves to study the effect of potassium humate solutions on the efficiency of photosynthesis (the maximum quantum yield of photosynthesis Fv / Fm).The efficiency of photosynthesis in photosystem II was determined using the formulas Fv = Fm - F0 and Fv / Fm. The chlorophyll fluorescence parameter Fv / Fm reflects the maximum quantum efficiency of photosystem II (PSII). Violations that occur under the influence of stress factors in the primary processes of photosynthesis are reflected in the change in chlorophyll fluorescence in plants. The index of maximum PSII quantum yield - Fv / Fm is used to estimate the maximum efficiency of PSII. This indicator provides information on the potential quantum efficiency of PSII. A decrease in this value may indicate the stress state of the plant and partial damage to PSII. (Kalaji et al., 2014, Delia Marcu et al., 2013).
Table 1: Some chemical characteristics of pine needle compost
X o. Total nitrogen % Humus % Nitrogen N/NH4 mg/kg Phosphorus mg/kg Potassium mg/kg K - according to soil gradation mg/kg Fe- according to soil gradation mg/kg N according to soil gradation mg/kg Absorbed Ca mq/ekv Absorbed Mg mq/ekv
7,0 0,422 9,6 70,55 277,5 376 450- 750 30-60 40-120 28,6 13,9
Figure 1. Influence of potassium humate (PH) on the growth and development of corn seedlings of the "Zagatala 68" variety obtained from seeds irradiated at a dose of 50 Gy
4 5 time
Figure 2. Influence of potassium humate (PH) on the growth and development of corn seedlings of the "Zagatala 68" variety obtained from seeds irradiated at a dose of 100 Gy
Figure 3. The effect of potassium humate solutions on the amount of chlorophyll pigments and carotenoids in corn
seedlings obtained from gamma-irradiated seeds of the "Zagatala 68" variety at a doses of 50 Gy and 100 Gy.
Figure 4. The effect of potassium humate solutions on the amount of malondialdehyde (mmol/g) in 100 Gy irradiated. 1-Cont, 2- Cont. +100 Gy, 3- Cont.+0,1% potassium humat, 4- Cont.+0,01% potassium humat
Figure 5. The effect of the potassium humate on the chlorophyll fluorescence parameter Fv / Fm in seedlings obtained from seeds irradiated at the dose 100Gy.
Based on the results of these studies, it can be said that when seeds are irradiated at a dose of 50 Gy, a significant effect of potassium humate solutions on the chlorophyll fluorescence index (Fv/Fm) in plants is not observed. When seeds are treated before irradiation with a 0.01% solution of potassium humate at a dose of 100 Gy, the chlorophyll fluorescence index in plants noticeably increases.
Thus, it can be said that the use of low doses of radiation and optimal concentrations of humate solutions and the creation of technologies on this basis makes it possible to grow agricultural plants under stressful conditions, and also increase plant productivity. In plants treated with 0.01% potassium humate solution, corncobs appeared earlier than others and were larger.
CONCLUSION
Based on these results, it can be concluded that
potassium humate solutions lead to decrease in the level of the product of lipid peroxidation -malondialdehyde and the maintenance of a normal concentration of photosynthetic pigments. The positive effect of potassium humate solutions was observed in the study of the development of seedlings obtained from irradiated corn seeds. 0.01% solution of potassium humate showed the highest antistress activity, reducing the harmful effects of gamma irradiation and salt stress.
CONFLICTS OF INTEREST
The authors declare that they have no potential conflicts of interest.
REFERENCES
Al-Rumaih, M.M., Al-Rumaih, M.M. (2008) Influence of ionizing radiation on antioxidant enzymes in three species of Trigonella. Am. J. Environ. Sci. 4, 151156.
Awwad, M.S.; K.S. El-Hedek, M.A. Bayoumi and T.A. Eid (2015) Effect of potassium humate appliction and irrigation water levels on maize yield, crop water productivity and some soil properties J.Soil Sci. and Agric. Eng., Mansoura Univ., 6(4), 461482.
Essam, Kandil, Nader R. Abdelsalam, Mansour A. Mansour, Hayssam M. Ali Manzer H. Siddiqui. (2020) Potentials of organic manure and potassium forms on maize (Zea mays L.) growth and production. Scientific Reports 10(1) DOI: https://doi.org/10.1038/s41598-020-65749-9
Holman, H. Y., Nieman, K., Sorensen, D. L., Miller, C. D., Martin, M. C., Borch, T., et al. (2002). Catalysis of PAH biodegradation by humic acid shown in synchrotron infrared studies. Environmental Science& Technology, 36, 1276-1280.
Kalaji H. M., Schansker G., Ladle R. J. et al. (2014) Frequently asked questions about in vivo chlorophyll fluorescence: practical issues. Photosynth. Res., 122. 121-158.
Kiong, A.L.P., Lai, A.G., Hussein, S., Harun, A.R. (2008) Physiological responses of Orthosiphon stamineus plantlets to gamma irradiation. Am-Eurasian. J. Sustain. Agric. 2, p.135-149
Ling, A.P.K., Chia, J.Y., Hussein, S., Harun, A.R. (2008) Physiological responses of Citrus sinesis to gamma irradiation. World Appl. Sci. 5, 12-19
Marcu Delia, Damian Grigore, Cosma Constantin, Cristea Victoria (2013) Gamma radiation effects on seed germination, growth and pigment content, and ESR study of induced free radicals in maize (Zea mays). J Biol Phys 39, 625-634 DOI 10.1007/s10867-013-9322-2013
Moghaddam, S.S., Jaafar, H., Ibrahim, R., Rahmat, A., Aziz, M.A., Philip, E. (2011) Effects of acute gamma irradiation on physiological traits and flavonoid accumulation of Centella asiatica. Molecules 16, 4994-5007
Montiller J.L., Cacas J.L. (2004) The upstream oxylipin profile of Arabidopsis thaliana: A tool to scan for oxidative stresses. Plant J. 40. 439-450
Murchie E. H., Lawson T. (2013). Chlorophyll
fuorescence analysis: A guide to good practice and understanding some new applications. J. Exp. Bot. 64(13). 3983-3998.
Muslimova Zohre, Azizov Ibrahim, Faracov Mahir.
(2013) Effect of ionizing radiation on pigment content and photochemical activity of chloroplasts in maize (Zea mays L.) leaves at partipication of humin comlexes. International conference Photosynthesis research for sustainability. June 59. Baku. Azerbaijan. p.111
Muslumova Z.H. (2015) influence of Metal Humic Complexes on Activity of Antioxidant System of y-irradiated Wheat Germs. International Journal of Secondary Metabolite, 2 (1), 13-17.
Qualls Robert G., Takiyama Akiko (2003)Formation and Loss of Humic Substances During Decomposition in a Pine Forest Floor. Soil Science Society of America Journal 67(3), 899-909 D0I:10.2136/sssaj2003.8990
Xavier de Campos S., Roney Resseti R. and Zittel R.
(2014) Monitoring and characterization of compost obtained from household waste and pine sawdust in a facultative reactor by conventional and spectroscopic analyses. Waste Management & Research, 32(12), 1186-1191 DOI: 10.1177/0734242X14543817
Stevenson, F.J. (1982) Humus Chemistry, Genesis, Composition, Reactions - New York: John Wiley&Son,. P - 443
Strid, A., Chow, W.S., Anderson, J.M.: (1990) Effects of supplementary gamma irradiation on photosynthesis in Pisum sativum. Biochem. 1020, p.260-268
Van Stempvoort, D. R., Lesage, S., Molson, J. (2005) The Use of Aqueous Humic Substances for In-Situ Remediation of Contaminated Aquifers. The Use of Humic Substances to Remediate Polluted Environments: From Theory to Practice, p.135-154.
Wei-Cai Zeng et al. (2012) Chemical composition, antioxidant, and antimicrobial activities of essential oil from pine needle (Cedrus deodara). J Food Sci.. 77(7):C824-9. doi: 10.1111/j.1750-3841
Zeng WC, Jia LR, Zhang Y, Cen JQ, Chen X, Gao H,
Feng S, Huang YN. Zeng WC, et al. (2011) Antibrowning and antimicrobial activities of the water-soluble extract from pine needles of Cedrus
deodara. J Food Microbiol. 153(1-2) 78-84. doi: 10.1016/j.ijfoodmicro.2011.10.019
