​Auxin-like and Cytokinin-like Effects of New Synthetic Pyrimidine Derivatives on the Growth and Photosynthesis of Wheat

The regulatory effect of new synthetic thienopyrimidine derivatives on the growth and photosynthesis of wheat (Triticum aestivum L.) variety Svitlana in the vegetative phase was studied. The regulatory effect of new synthetic thienopyrimidine derivatives was compared with the regulatory effect of auxin IAA (1H-indol-3-yl)acetic acid) or synthetic plant growth regulators Methyur (sodium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine) and Kamethur (potassium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine). After 2 weeks, morphometric parameters (such as average length of shoots and roots (mm), average biomass of 10 plants (g)) and biochemical parameters (such as content of photosynthetic pigments (µg/ml)) of wheat plants grown from seeds treated with synthetic thienopyrimidine derivatives, or auxin IAA, or synthetic plant growth regulators Methyur and Kamethur at a concentration of 10^-6M, were measured and compared with similar parameters of control wheat plants grown from seeds treated with distilled water. The regulatory effect of new synthetic thienopyrimidine derivatives on the morphometric and biochemical parameters of wheat plants was similar or higher compared to the regulatory effect of auxin IAA, or synthetic plant growth regulators Methyur and Kamethur. The relationship between the chemical structure of new synthetic thienopyrimidine derivatives and their regulatory effect on the growth and photosynthesis of wheat plants was revealed. The most biologically active thienopyrimidine derivatives are proposed to be used as new synthetic physiological analogues of auxins and cytokinins to improve growth and increase photosynthesis of wheat (Triticum aestivum L.) variety Svitlana in the vegetative phase.

As is known, phytohormones auxins and cytokinins are key regulators of plant growth and development, which are synthesized in the apical meristems of shoots and roots, young leaves, seeds, and fruits [1-4]. They exhibit a stimulating effect on seed germination, the formation and growth of shoots, and adventitious and lateral roots of plants in the vegetative stage [1-4]. Considerable attention of plant biologists is devoted to the screening of new effective analogues of auxins and cytokinins of synthetic origin for their use in agriculture to improve growth and increase the productivity of agricultural crops. In recent years, new synthetic analogues of auxins and cytokinins have been created, such as NAA (1-naphthylacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), 3,4-D (3,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), 4-CPA (4-chloro-pheno xyacetic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid), picloram (4-amino-3,5,6-trichloropyridine-2-carboxylic acid), kinetin (6-furfurylaminopurine), 2iP (N6-(2-isopentenyl) adenine), BA (N6-benzyladenine), BAP (6-benzylaminopurine), BPA (N-benzyl-9-(2-tetrahydropyranyl)-adenine), tetrahydro pyranyl-benzyladenine (PBA), TDS (thidiazuron), that have a physiological effect similar to natural phytohormones such as IAA (indole-3-acetic acid), 4-Cl-IAA (4-chloro-IAA), PAA (phenylacetic acid), IBA (indole-3-butyric acid), IPA (indole-3-pyruvic acid), 2-(2,4-dichloro-phenoxy) propionic acid (2,4-DP), indole-3-lactic acid (ILA), zeatin (N6-(4-Hydroxy-3-methyl-2-buten-1-yl)adenine) on the growth and development of plants during ontogenesis, due to which they are used in agriculture as plant growth regulators [3-13].

Our previously conducted studies show that the synthetic plant growth regulators Methyur (sodium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine), Kamethur (potassium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine), and new synthetic pyrimidine derivatives when used in concentrations from 10-5M to 10-8M, demonstrate auxin-like and cytokinin-like effects on growth and development, as well as on the productivity of major grain, leguminous, vegetable, industrial and horticultural crops [14-22].

Currently, new synthetic compounds belonging to thienopyrimidine derivatives are used in medicine as therapeutic agents, showing antibacterial, antifungal, antiviral, anticancer, antioxidant, anti-inflammatory, antitubercular, antidiabetic, antihypertensive, cardiotonic, anticonvulsant, antimalarial, antihelminthic and analgesic activities through inhibition of various enzymes and pathways [23-30]. Besides this, a very promising approach is the screening of new synthetic compounds among thienopyrimidine derivatives that can be practically used in agriculture as plant growth regulators, herbicides, pesticides, and insecticides by inhibiting various enzymes of weeds and insects [31-39].

Our present work is aimed at the screening of new synthetic compounds among thienopyrimidine derivatives, which show the ability to demonstrate auxin-like and cytokinin-like effects on the growth and photosynthesis of an important agricultural crop - wheat (Triticum aestivum L.) variety Svitlana.

Synthetic plant growth regulators Methyur (sodium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine), Kamethur (potassium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine and new synthetic thienopyrimidine derivatives (compounds № 1 – 11) were synthesized at the Department for Chemistry of Bioactive Nitrogen-Containing Heterocyclic Compounds, V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine. Auxin IAA (1H-indol-3-yl)acetic acid was manufactured by Sigma-Aldrich, USA (Table 1).

Plant growth conditions

The seeds of wheat (Triticum aestivum L.) variety Svitlana were sterilized with 1% KMnO4 solution for 15 min, then treated with 96% ethanol solution for 1 min, after which they were washed three times with sterile distilled water. After this procedure, wheat seeds were placed in the plastic cuvettes (each containing 20 seeds - 25 seeds) on the perlite moistened with distilled water (control sample) or water solutions of auxin IAA, or synthetic plant growth regulators Methyur and Kamethur, or synthetic thienopyrimidine derivatives used in the most physiologically active concentration of 10^-6M (experimental samples). Then the wheat seeds were placed in a thermostat for germination in the dark at a temperature of 20 °C - 22 °C for 48 hours. After the appearance of wheat seedlings, they were placed in a climate chamber, where they were grown for 2 weeks at 16/8 h light/dark conditions, at the temperature of 20 °C - 22 °C, light intensity of 3000 lux, and air humidity of 60% - 80%. Comparative analysis of morphometric parameters of wheat plants (average length of shoots and roots (mm), average biomass of 10 plants (g)) was carried out at the end of the two-week period according to the method [40].

Determination of the content of photosynthetic pigments

To perform the extraction of photosynthetic pigments, we homogenized a sample (500 mg) of wheat leaves in the porcelain mortar cooled at the temperature of 10 °С 96% ethanol at the ratio of 1: 10 (weight: volume) with the addition of 0,1 - 0,2 g CaCO3 (to neutralize the plant acids). The 1 ml of obtained homogenate was centrifuged at 8000 g in a refrigerated centrifuge K24D (MLW, Engelsdorf, Germany) for 5 min at the temperature of 4 °С. The obtained precipitate was washed three times, with 1 ml 96% ethanol, and centrifuged at the above-mentioned conditions. After this procedure, the optical density of chlorophyll a, chlorophyll b, and carotenoid in the obtained extract was measured using a spectrophotometer Specord M-40 (Carl Zeiss, Germany).

The content of chlorophyll a, chlorophyll b, and carotenoids in wheat leaves was calculated in accordance with formula [41,42]:

Cchl a = 13.36 × A664.2 – 5.19 × A648.6,

Cchl b = 27.43 × A648.6 – 8.12A × 664.2,

Cchl (a + b) = 5.24 × A664.2 + 22.24 × A648.6,

Ccar = (1000 × A470 – 2.13 × Cchl a – 97.64 × Cchlb)/209,

Where,

Cchl – concentration of chlorophylls (µg/ml), Cchl a – concentration of chlorophyll a (µg/ml), Cchl b – concentration of chlorophyll b (µg/ml), Ccar – concentration of carotenoids (µg/ml), А – absorbance value at a proper wavelength in nm.

The chlorophyll and carotenoids content per 1 g of Fresh Weight (FW) extracted from wheat leaves was calculated by the following formula (separately for chlorophyll a, chlorophyll b, and carotenoids):

A1 = (C × V)/(1000 × a1),

Where, A1 – content of chlorophyll a, chlorophyll b, or carotenoids (mg/g FW),

C - Concentration of pigments (µg/ml),

V - Volume of extract (ml),

a1 - Sample of wheat leaves (g).

The content of chlorophyll a, chlorophyll b, and carotenoids (%) determined in experimental wheat leaves was calculated according to similar parameters determined in control wheat leaves.

Statistical data analysis

Each experiment was performed three times. Statistical processing of the experimental data was carried out using Student’s t-test with a significance level of p ≤ 0.05; mean values ± standard deviation (± SD) [43].

The regulatory effect of new synthetic thienopyrimidine derivatives compared to the regulatory effect of auxin IAA and synthetic plant growth regulators Methyur and Kamethur on the vegetative growth of wheat (Triticum aestivum L.) variety Svitlana was studied. The morphometric parameters of wheat plants grown from seeds treated with synthetic thienopyrimidine derivatives (compounds № 1 – 11, Table 1), auxin IAA, and synthetic plant growth regulators Methyur and Kamethur at a concentration of 10^-6M, measured after 2 weeks were compared with similar parameters of control wheat plants grown from seeds treated with distilled water. The obtained results show that the growth-regulatory effect of new synthetic thienopyrimidine derivatives was similar to the growth-regulatory effect of auxin IAA or synthetic plant growth regulators Methyur and Kamethur (Figure 1).

Increased growth and development of shoots and roots of wheat plants was observed for 2 weeks compared to control plants (Figure 1).

The parameters of the average length of shoots of 2-week-old wheat plants grown from seeds treated with auxin IAA at a concentration of 10^-6M, statistically significantly increased by 32,67%, respectively, compared to control plants (Figure 2).

The parameters of the average length of shoots of 2-week-old wheat plants grown from seeds treated with plant growth regulator Methyur at a concentration of 10^-6M, statistically significantly increased by 49,33%, respectively, compared to control plants (Figure 2). The parameters of the average length of shoots of 2-week-old wheat plants grown from seeds treated with plant growth regulator Kamethur at a concentration of 10^-6M, statistically significantly increased by 46,67%, respectively, compared to control plants (Figure 2). The parameters of the average length of shoots of 2-week-old wheat plants grown from seeds treated with the most active synthetic thienopyrimidine derivatives № 2, 3, 5, 7, 9 and 11 at a concentration of 10^-6M, statistically significantly increased by 33,33% – 45,33%, respectively, compared to control plants (Figure 2). The parameters of the average length of shoots of 2-week-old wheat plants grown from seeds treated with the less active synthetic thienopyrimidine derivatives № 4, 6, 8 and 10 at a concentration of 10^-6M, statistically significantly increased by 24% - 28%, respectively, compared to control plants (Figure 2). The parameters of the average length of shoots of 2-week-old wheat plants grown from seeds treated with the less active synthetic thienopyrimidine derivative № 1 at a concentration of 10^-6M, increased by 16,67%, but did not differ statistically significantly from control plants (Figure 2).

The parameters of the average length of roots of 2-week-old wheat plants grown from seeds treated with auxin IAA at a concentration of 10^-6M, statistically significantly increased by 75,51%, respectively, compared to control plants (Figure 3). The parameters of the average length of roots of 2-week-old wheat plants grown from seeds treated with plant growth regulator Methyur at a concentration of 10^-6M, statistically significantly increased by 255,1%, respectively, compared to control plants (Figure 3). The parameters of the average length of roots of 2-week-old wheat plants grown from seeds treated with plant growth regulator Kamethur at a concentration of 10^-6M, statistically significantly increased by 132,65%, respectively, compared to control plants (Figure 3). The parameters of the average length of roots of 2-week-old wheat plants grown from seeds treated with the most active synthetic thienopyrimidine derivatives № 2 – 4, 6, 7, 9 and 11 at a concentration of 10^-6M, statistically significantly increased by 95,92% – 177,55%, respectively, compared to control plants (Figure 3). The parameters of the average length of roots of 2-week-old wheat plants grown from seeds treated with the less active synthetic thienopyrimidine derivatives № 1, 5, 8 and 10 at a concentration of 10^-6M, statistically significantly increased by 48,98% – 75,51 %, respectively, compared to control plants (Figure 3).

The parameters of the average biomass of 10 plants (g) of 2-week-old wheat plants grown from seeds treated with auxin IAA at a concentration of 10^-6M, statistically significantly increased by 35,16%, respectively, compared to control plants (Figure 4). The parameters of the average biomass of 10 plants (g) of 2-week-old wheat plants grown from seeds treated with plant growth regulator Methyur at a concentration of 10^-6M, statistically significantly increased by 81,31%, respectively, compared to control plants (Figure 4). The parameters of the average biomass of 10 plants (g) of 2-week-old wheat plants grown from seeds treated with plant growth regulator Kamethur at a concentration of 10^-6M, statistically significantly increased by 71,43%, respectively, compared to control plants (Figure 4). The parameters of the average biomass of 10 plants (g) of 2-week-old wheat plants grown from seeds treated with the most active synthetic thienopyrimidine derivatives № 3, 6, 7, 9 and 11 at a concentration of 10^-6M, statistically significantly increased by 46,15% – 93,41%, respectively, compared to control plants (Figure 4). The parameters of the average biomass of 10 plants (g) of 2-week-old wheat plants grown from seeds treated with the less active synthetic thienopyrimidine derivatives № 1, 2 and 4, at a concentration of 10^-6M, statistically significantly increased by 34,07% – 42,86%, respectively, compared to control plants (Figure 4). The parameters of the average biomass of 10 plants (g) of 2-week-old wheat plants grown from seeds treated with the less active synthetic thienopyrimidine derivatives № 5, 8 and 10 at a concentration of 10^-6M, increased by 14,29% – 20,88%, but did not differ statistically significantly from control plants (Figure 4).

Summarizing the obtained morphometric parameters of wheat plants (average length of shoots and roots (mm), average biomass of 10 plants (g)), it should be noted that new synthetic thienopyrimidine derivatives (compounds № 2, 3, 6, 7, 9 and 11) showed the highest growth-regulatory effect, which was similar or higher than that of the auxin IAA or the synthetic plant growth regulators Methyur and Kamethur.

The regulatory effect of new synthetic thienopyrimidine derivatives (compounds № 1 – 11, Table 1), auxin IAA, and synthetic plant growth regulators Methyur and Kamethur at a concentration of 10^-6M on the content of photosynthetic pigments (chlorophyll a, chlorophyll b, chlorophylls a+b, and carotenoids) in the leaves of 2-week-old wheat (Triticum aestivum L.) variety Svitlana was also studied. The content of photosynthetic pigments in the leaves of 2-week-old wheat plants grown from seeds treated with auxin IAA at a concentration of 10^-6M, statistically significantly increased: chlorophyll a – by 67,72%, chlorophyll b - by 89,7%, chlorophylls a+b - by 73,7%, carotenoids - by 56,7%, respectively, compared to control plants (Figure 5). The content of photosynthetic pigments in the leaves of 2-week-old wheat plants grown from seeds treated with plant growth regulator Methyur at a concentration of 10^-6M, statistically significantly increased: chlorophyll a – by 95,56 %, chlorophyll b - by 183,22%, chlorophylls a+b - by 119,42%, carotenoids - by 47,9%, respectively, compared to control plants (Figure 5). The content of photosynthetic pigments in the leaves of 2-week-old wheat plants grown from seeds treated with plant growth regulator Kamethur at a concentration of 10^-6M, statistically significantly increased: chlorophyll a - by 54,25%, chlorophyll b - by 293,63%, chlorophylls a+b - by 119,42%, respectively, compared to control plants (Figure 5). The content of photosynthetic pigments in the leaves of 2-week-old wheat plants grown from seeds treated with the most active synthetic thienopyrimidine derivatives № 2 – 8 at a concentration of 10^-6M, statistically significantly increased: chlorophyll a - by 51,54% – 95,21%, chlorophyll b - by 45,29% – 160,8%, chlorophylls a+b - by 49,84% – 113,07%, carotenoids - by 23,3% – 108,38%, respectively, compared to control plants (Figure 5). The content of photosynthetic pigments in the leaves of 2-week-old wheat plants grown

from seeds treated with the less active synthetic thienopyrimidine derivatives № 1, 9, 10 and 11 at a concentration of 10^-6M, statistically significantly increased: chlorophyll a – by 19,43% – 35,49%, chlorophyll b – by 37,79% – 70,61%, chlorophylls a+b - by 26,72% – 36,67%, respectively, compared to control plants (Figure 5). The content of carotenoids in the leaves of 2-week-old wheat plants grown from seeds treated with synthetic thienopyrimidine derivative № 1 at a concentration of 10^-6M, statistically significantly increased by 24,88%, respectively, compared to control plants (Figure 5). At the same time, the content of carotenoids in the leaves of 2-week-old wheat plants grown from seeds treated with plant growth regulator Kamethur and synthetic thienopyrimidine derivatives № 9, 10 and 11 at a concentration of 10^-6M, did not differ statistically significantly from control plants (Figure 5).

Thus, the obtained results confirmed the positive regulatory effect of synthetic thienopyrimidine derivatives (compounds № 2 – 8) at a concentration of 10^-6M on increasing the content of chlorophylls a, b, and carotenoids (µg/ml) in the leaves of 2-week-old wheat (Triticum aestivum L.) variety Svitlana, which play a key role in photosynthesis and ensuring plant productivity [41,42]. The regulatory effect of synthetic thienopyrimidine derivatives (compounds № 2 – 8) was similar to or higher than that of the auxin IAA or the synthetic plant growth regulators Methyur and Kamethur.

Analyzing the relationship between the chemical structure and biological activity of new most active synthetic thienopyrimidine derivatives № 2, 3, 5 – 9 and 11, it can be assumed that the high growth-regulatory effect of these compounds is associated with the presence of substituents in their chemical structure (Table 1): compound № 2 contains an allyl substituent in position 3, a phenylsulfonyl group in position 5 of the 2-thioxo-2,3-dihydro-1H-pyrimidin-4-one ring; compound № 3 contains a benzyl substituent in position 5, a methyl group in position 6 of the 2-thioxo-2,3-dihydro-1H-pyrimidin-4-one ring; compound № 5 contains a p-tolyl group in position 6, a cyano group in position 5 of the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine ring; compound № 6 contains a phenyl group in position 6, a cyano group in position 5 of the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine ring; compound № 7 contains a methylsulfanyl group in position 2, a p-tolyl group in position 4, and a cyano group in position 5 of the 6-oxo-1,6-dihydropyrimidine ring; compound № 8 contains a methyl group in position 6, a phenyl group in position 4, and an ethoxycarbonyl group in position 5 of the 2-thioxo-1,2,3,4-tetrahydropyrimidine ring; compound № 9 contains a methyl group in position 6, a 4-methoxyphenyl group in position 4, and an ethoxycarbonyl group in position 5 of the 2-thioxo-1,2,3,4-tetrahydropyrimidine ring; compound № 11 contains a methyl group in position 6, a 4-hydroxyphenyl group in position 4, and an ethoxycarbonyl group in position 5 of the 2-thioxo-1,2,3,4-tetrahydropyrimidine ring.

At the same time, the decrease in the growth-regulatory effect of synthetic thienopyrimidine derivatives № 1, 4, and 10 can be explained by the presence of substituents in the chemical structures of these compounds (Table 1): compound № 1 contains a benzenesulfonyl group in position 5, an ethyl group in position 3 of the 2-thioxo-2,3-dihydro-1H-pyrimidin-4-one ring; compound № 4 contains a phenyl group in position 3, a benzenesulfonyl group in position 5 of the 2-thioxo-2,3-dihydro-1H-pyrimidin-4-one ring; compound № 10 contains a methyl group in position 6, a 4-methoxycarbonylphenyl group in position 4, and an ethoxycarbonyl group in position 5 of the 2-thioxo-1,2,3,4-tetrahydropyrimidine ring.

Summarizing the obtained morphometric and biochemical parameters of wheat plants, it should be noted that the regulatory effect of the new most active synthetic thienopyrimidine derivatives № 2, 3, 5 – 9, and 11 were similar or higher than that of the auxin IAA or the synthetic plant growth regulators Methyur and Kamethur.

Based on data from studying the physiological and molecular mechanisms of signal transduction of natural auxins and cytokinins and their synthetic physiological analogues, it can be assumed that the growth-regulating effect of thienopyrimidine derivatives is explained by both their influence on the pathways of perception and transmission of natural auxin and cytokinin signals and their influence on the pathways of biosynthesis, metabolism, and transport of natural auxins and cytokinins in plant cells, which regulate the growth and development of plant shoots and roots and also slow down the degradation of chlorophylls and carotenoids in plant cells [1-13,44-60].

The obtained results confirmed the possibility of practical use of the most active thienopyrimidine derivatives (compounds № 2, 3, 5 – 9, and 11) at a concentration of 10^-6M as new synthetic physiological analogues of auxins and cytokinins for the regulation of growth and development of wheat (Triticum aestivum L.) variety Svitlana in the vegetative phase and increasing the content of photosynthetic pigments in plant leaves, which ensure plant productivity.

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