THE EFFECT OF SALT STRESS AND BIOSTIMULANTS BASED ON GLYCYRRHIZIC ACID ON COTTON PHOTOSYNTHETIC PIGMENTS

ABSTRACT

Changes in the number of photosynthetic pigments among physiological parameters can be considered as a reliable indicator in the assessment of salt resistance of cotton varieties. Quantitative changes in photosynthetic pigments under the influence of three different concentrations of NaCl (50 mM, 100 mM, 200 mM) in the cotton variety (Sultan) were detected. With an increase in the level of salt stress, the amount of chlorophyll decreased and the number of carotenoids increased in the leaves of 14 day-old cotton seedlings. We investigated the use of cotton growing and assessed the benefits of foliar treatment with newly developed preparations based on glycyrrhizic acid - preparation DAG-1, copper diglycyrrhizinate ((GK)₂Cu), zinc diglycyrrhizinate (Zn(GK)₂). The results showed the effectiveness of the DAG-1 stimulant under salinity conditions in which the pigments of photosynthesis were increased, while the effect of Cu(GA)₂ and Zn(GA)₂ did not differ significantly from the control.

АННОТАЦИЯ

Изменения количества фотосинтетических пигментов среди физиологических параметров можно рассматривать как надежный индикатор при оценке солеустойчивости сортов хлопчатника. Выявлены количественные изменения фотосинтетических пигментов под влиянием трех различных концентраций NaCl (50 мМ, 100 мМ, 200 мМ) у сорта хлопчатник Султан. С увеличением уровня солевого стресса количество хлорофилла уменьшалось, а количество каротиноидов увеличивалось в листьях 14-дневных проростков хлопчатника. Мы исследовали возможности выращивания хлопка и оценили преимущества внекорневой обработки новыми препаратами на основе глицирризиновой кислоты - препаратом ДАГ-1, диглицирризинатом меди ((GK)₂Cu), диглицирризинатом цинка (Zn(GK)₂). Результаты показали эффективность стимулятора ДАГ-1 в условиях засоления, при которых пигменты фотосинтеза были увеличены, в то время как действие (GK)₂Cu и Zn (GK)₂ существенно не отличалось от контроля.

Keywords: cotton, saline stress, photosynthesis, chlorophyll, carotenoids, glycyrrhizic acid.

Ключевые слова: хлопчатник, солевой стресс, фотосинтез, хлорофилл, каротиноиды, глицирризиновая кислота.

Salt stress is one of the major abiotic factors that limiting the productivity of agricultural plants and negatively affecting their germination and viability [1]. Salinity is a serious threat for cot-ton growth, yield and fiber quality. Therefore, understanding of cotton response to salinity, its resistance mechanism and looking into management techniques may assist in formulating strategies to improve cotton performance under saline conditions.

Salt stress imposes specific ions toxicity, somatically induced water stress and nutrients imbalance which imparts adverse effects on plant growth, development and ultimately crop establishment [2]. It is known that the photosynthetic system of plants is very sensitive to the effects of high temperature and salts [3]. A decrease in the rate of photosynthesis in many plant species is a clear indication of salt stress. It has been reported that salt stress affects the process of photosynthesis due to disorientation of the chloroplast lamellar system and loss of chloroplast integrity, leading to a decrease in photosystem activity [4-6]. Therefore, changes in the composition of photosynthetic pigments among physiological parameters can be considered as a reliable indicator in the assessment of salt resistance of varieties [7].

Carotenoids are considered to be another important pigment of the photosynthetic mechanism involved in the accumulation of light energy during photosynthesis. Decreased expression of genes responsible for the biosynthesis of carotenoids during salt stress in plants is associated with a slowing of the rate of photosynthesis, resulting in decreased productivity [5]. Rafique et al [8] reported that carotenoid contents degradation is slower as compared to chlorophyll in salinity stress. In particular, the indicators of the photosynthetic apparatus of plants are informative, and the amount of pigment in the tissues determines their functional state, changes that occur during growth, development and stress.

The study of the mechanisms of salinity resistance of plants is one of the current theoretical and scientific problems, with a deep emphasis on the development of methods for the use of physiologically active substances in increasing the resistance of plants to salt stress and their widespread application in agricultural production. The use of plant biostimulants on crop plants can generate multiple benefits with reported effects including enhanced rooting, higher crop and fruit yields, drought and salt tolerance, enhanced photo-synthetic activity [9]. Plant biostimulants include diverse formulations of compounds, substances and other products, such as microorganisms, trace elements, enzymes, plant growth regulators that are applied to plants to regulate and enhance the crop’s physiological processes, thus making them more efficient. Consequently, the products can enhance nutrient availability, waterholding capacity, increase antioxidants, enhance metabolism and increase chlorophyll production in plants [10].

This study aimed to investigate the effects of salt stress on photosynthetic pigments of cotton leaves and to determine the effectiveness of leaf treatment with stimulant based on glycyrrhizic acid and its complex compounds to increase the resistance of cotton to salinity. The use of triterpene glycosides to assemble compxes with biologically active compounds is becoming more and more popular for the development of new transportable forms of low-dose preparations. Glycyrrhizic acid (GA) is the dominant triterpene glycoside from licorice (Glycyrrhiza glabra L.) roots. GA can act as a plant growth regulator, which is characteristic of phytohormones. GA at a concentration of 10^–6 M caused weak cytokinin-like activity in amaranth sprouts [11]. High biological activity of individual GA and SA prompted the development of a method to enable practical use of the supramolecular complex in DAG-1 for agricultural purposes as a growth regulator and resistance inductor in cotton growing in saline soils. The glycyrrhizic acid copper – Cu(GA)₂, and zinc – Zn(GA)₂ complexes are also been synthesized.

Materials and methods

Seeds of cotton G. hirzutum cultivars Sultan was provided by the Scientific Research Institute of Cotton Breeding, Seed Production and Cultivation Agrotechnology of the Republic of Uzbekistan. Stimulant DAG-1, copper and zinc glycyrrhizinates were synthesized by Institute of Bioorganic Chemistry of Academy Sciences of Republic Uzbekistan. The studies were conducted in the laboratory and the field conditions. Under laboratory conditions, the seeds were wrapped in filter paper, placed as controls in aqueous and NaCl solutions of three different concentrations (50 mM, 100 mM, 200 mM) and grown for 14 days at a temperature of 25⁰C in light and 17⁰C in dark conditions. In randomized field experiments, Sultan cotton seedlings in saline soils of Bayovut district of Syrdarya region were treated separately with leaves with DAG-1, glycyrrhizic acid copper – Cu(GA)₂, and zinc – Zn(GA)₂ complexes at a concentration of 10^-7 M during six leaf stages and the number of photosynthetic pigments in the leaves was analyzed on day 3.

Isolation and chlorophyll determination. The chlorophyll concentration was determined in 80% acetone extract [12]. Briefly, 0.2 g of the sample was extracted in 80% cold acetone. The optical densities of total chlorophyll (652 nm), chlorophyll a (663 nm), chlorophyll b (645 nm), and total carotenoids (470 nm) in the extracts were determined on a UV-1800 spectrophotometer (Shimadzu, Japan). The amount of chlorophyll and carotenoids was calculated using Lichtenthaler and Welburn formulas [13].

The results were processed using Excel. The mean deviation index (± M) and statistical reliability index (P) were determined, and results less than P <0.05 were considered statistically significant.

Results and discussion

The results showed that with increasing saline stress levels the chlorophyll content was decreased and the number of carotenoids increased in 7-day old cotton seedlings (Table 1). The total chlorophyll content of the cotton seedlings at the salinity concentration of 50 mM, 100 mM, 200 mM decreased by 8.4%, 21%, 41.6% respectively.

Table 1.

Effect of salinity on photosynthetic pigments of cotton leave tissues

 (mg/g FW)

When studying the amount of Chl a and Chl b, which are the main indicators of the photosynthetic system, Chl a compared to the control in cotton seedlings grown in solutions with a concentration of 50 mM, 100 mM, 200 mM was found to de-crease by 4.6%, 6.1%, 39.1% respectively, Chl b decrease by 22.6%, 46.5%, and 36.7%, respectively. These results showed that the amount of Chl b was significantly reduced under the influence of salt relative to the amount of Chl a. It was found that the amount of carotenoids increased by 43.6%, 60.6%, and 80.7%, respectively, with increasing salinity.

Inhibition of photosynthesis under the influence of salt may be due in part to the closure of leaf openings, although there are also data on the direct effect of salt on certain biochemical and photochemical processes [15]. According to analyzes, NaCl concentrations of 100 and 200 mM reduced the quantitative values of photosynthetic pigments (Chl a and b). Meng et al. [16] reported that a decrease in photosynthesis is associated with a reduction in chlorophyll contents and alteration in ultrastructure of chlorophyll. Significant reduction in chlorophyll contents (a and b) was observed in cotton cultivars with the in-crease in salinity level [17]. This may be due to inhibition of the activity of specific enzymes involved in chlorophyll synthesis.

The number of carotenoids was found to increase by 43.6%, 60.6%, and 80.7%, respectively, with in-creasing salinity. At different salinity levels, an increase in carotenoids, anthocyanins and flavones contents were recorded while at the same time other photosynthetic pigments i.e. Chl a and b were drastically de-creased. Since there was no statistically significant difference between the samples grown in 50 and 100 mM concentrated solutions of NaCl relative to the control, the 100 mM concentration of NaCl was conditionally set as the tolerance limit to the saline environment for the Sultan variety of cotton.

In field experiments, Sultan cotton seedlings in saline soils of Buyout district of Sirdarya region were treated separately with leaves with DAG-1, glycyrrhizic acid copper – Cu(GA)₂, and zinc – Zn(GA)₂ complexes at a concentration of 10^-7 M during six leaf stages and the number of photosynthetic pigments in the leaves was analyzed on day 3 (Table 2).

Table 2.

The content photosynthetic pigments of cotton leave tissues during foliar treatment with DAG-1, Cu(GA)₂, and Zn(GA)₂ (mg/g FW)

According to obtained results, the content of photosynthetic pigments in cotton leaves: total chlorophyll content, Chl a, Chl b, and carotenoids was found to be higher than the control in samples treated with DAG-1. The stimulant DAG-1 mainly stimulated an increase in total chlorophyll (a and b), Chl a, and carotenoids.

Specific response reactions in photosynthetic pigments were observed as a result of the treatment of cotton with leaves with copper and zinc diglycyrrhizinates. In samples treated with Cu(GA)₂ total chlorophyll was reduced by 14.43%, Chl a by 27.1%, carotenoids by 12.4%, and Chl b by 8.26% compared to controls. In samples treated with Zn(GA)₂ although total chlorophyll content increased by 6.71% compared to the control, Chl a decreased by 37.07%, Chl b by 42.15%, and carotenoids by 10.76%. Analysis of the results showed that under soil salinity, Cu(GA)₂ increased the amount of Chl b in cotton leaves by 8.26%, and (GA)₂Cu increased the total chlorophyll content by 6.71%. It is known that short-wave pigment forms in plants not only collect light [18], but also perform a protective function that prevents the oxidation of chlorophylls during electron transfer in the presence of transient metals [19]. In our experiments, a significant decrease in carotenoids under the influence of copper and zinc diglycyrrhizinate may be due to their oxidation and, consequently, a protective antioxidant effect.

It is supposed that the integrity of plastids is maintained due to the activity of antioxidant systems that reduce the number of reactive oxygen species and resulting in increased photosynthetic activity in cotton leaf samples treated with DAG-1.

The results showed the effectiveness of the DAG-1 stimulant under salinity conditions in which the pigments of photosynthesis were increased, while the effect of Cu(GA)₂ and Zn(GA)₂ did not differ significantly from the control.

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