Wheat (Triticum aestivum L.) Reaction to New Bifunctional Carbamate Compounds

Extreme environmental conditions increase the risk of abiotic stresses in plants, which reduce productivity of land. The investigation and developmentof synthetic approach to new antistress compounds, increasing the resistance of plants to negative factors, are relevant to the present. The objective of this study was to synthesize and to test the effect of a series of new carbamate and oxamate plant growth regulators on early vegetation processes of wheat (Triticum aestivum L.). Three independent series of experiments were conducted using lipophilic compounds N-(2,4-Dimethylphenyl)-N’-i-propoxycarbonylaminoethylurea (1) and O-Isopropyl-N-[2-(4-methylphenylaminocarbonyloxy)etyl]oxamate (2) and water-soluble compounds O-i-Propyl-N-(2-hydroxyethylamino)carbamate (3) and O-i-Propyl-N-(2-hydroxyethyl)oxamate (4). Strong stimulating effect on shoots and roots growth in the wheat was found for all studied compounds in all experiments. The highest germination rate (89.6-96.3%) was registered for compound 4. The use of the studied substances 1, 3 and 4 in all cases leads to significant increase in the Seedling Vigor Index (SVI). The high energy index of compounds 3 and 4 can lead to increased photosynthesis and, as a result, to an increase in crop yield. Based on the data obtained on the values of relative water content (RWC) indicators and their components, it can be assumed that all plants after treatment with substances 1-4 are able to tolerate unfavorable weather phenomena. Experimental results show that the percentage of plant recovery after resuming watering was 84-100%. Thus, the data obtained indicate that the synthesized compounds exhibit antistress and growth regulatory activity.


Introduction
The global change of environment is one of the main problems of the last century (Raza et al., 2019) and, according to forecasts of the Food and Agriculture Organization of the United Nations, by the end of this century, along with the increasing average temperature, crop production will be dramatically decreasing worldwide (Ito et al., 2018;Rogelj et al., 2016). Indeed, continuous droughts, heavy rains, temperature fluctuations, soil salinization and insect pests drastically lower agricultural productivity (Dhankher & Foyer, 2018). The use of plant growth regulators improves resistance to extreme conditions and mitigates the effects of stress on plants (Chauhan et al., 2019;Sosnowski et al., 2017).
Wheat is the main grain crop, occupying about 40% of agricultural areas in the world (FAOSTAT, 2017) and its production is directly dependent on the environment conditions (Asseng et al., 2015). In this regard, drought and sharp temperature changes are the key stress factors affecting crop productivity (Hatfield & Prueger, 2015): strong temperature drop leads to sterility, while dehydration adversely affects the morphology and physiology of plants (Barlow et al., 2015;Salehi-Lisar & Bakhshayeshan-Agdam, 2016). Unfavorable environmental conditions increase the risk of abiotic stresses in plants ( Thornton et al., 2014) and reduce productivity of land (Tebaldi & Lobell, 2018). The investigation and design of new antistress drugs, increasing the resistance of plants to negative factors, are quite relevant today.
In order to overcome the consequences of global environmental changes, farmers diversify crop rotation (Peltonen-Sainio et al., 2020), use insecticides, fungicides and plant growth regulators (PGR), which include chlormequat chloride (CCC), affecting the height of plants, lodging and yield of crops (Finch et al., 2014;Brinkman et al., 2014). In Russia, efforts to create plant growth regulators were initiated as early as the in the 1970's. Over the past years, synthetic PGR with a wide range of biological activity have been created, enhancing the productivity of cultivated plants due to the structural similarity to natural plant hormones, which increases their resistance to a complex of adverse environmental factors (Razina et al., 2018;Oshchepkov et al., 2020).
One of such PGR is Cartolin-2 or N-(isopropoxycarbonyl)-O-(4-chlorophenylcarbamoyl)ethanolamine, a compound with antistress activity that enhances the resistance of plants, particularly wheat, to various adverse conditions: drought, low temperatures, salinization and pathogenic fungi. It accelerates plant growth and the progression of phenophases and promotes biomass accumulation through the activation of protein, carbohydratesand chlorophyll biosynthesis. During droughts it has a protective effect on the photosynthetic apparatus of plant leaves, increases the activity of RNA polymerase and the fraction of the polysome in the ribosom complex (Shapovalov & Zubkova, 2003). However, the synthesis methodof this compound includes the use of toxic phosgene (Patent SU, 1992), which imposes serious restrictions on the organization of its production.
Earlier we reported  that substances structurally similar to compound 1 demonstrate a protective effect against herbicides with hormonal and antimetabolic mechanisms of action and, therefore, they can be used to increase the selectivity of herbicides and for growing crops on soils contaminated with herbicides. Therefore, intensive investigation and testing of biological activity of new analogues of PGR, whose synthesis method is safer and easier to implement technologically  are in high demand. Previously, for compounds of the same class, we carried out preliminary tests on tobacco cell culture (growth tests under controlled conditions) and showed that tested compounds have an evident, although multidirectional effect on the metabolic processes (Kovalenko et al., 2020). The objective of this study was to synthesize and test the activity of new carbamate and oxamate plant growth regulators on early vegetation processes of wheat (Triticum aestivum L.).

Materials and Methods
The experiments were conducted at the laboratories of the Nesmeyanov Institute of Organoelement Compounds of Russian Academy Sciences, Moscow, Russia from September 2020 to August 2021. The geographic coordinates of the site are 55°42′0.3″N; 37°34′30″E.

Synthesis of Target Plant Growth Regulators 1-4
The synthesis of carbamates (1, 2) and oxamates (3, 4) was carried out at the Department of Chemistry and Technology of Biomedical Preparations of Mendeleev University of Chemical Technology of Russia (55°46′44″N; 37°35′43″E), Figure 1. The application of compounds 3, 4 is determined by their good solubility in water and ease of penetration through cell membranes. Compounds 1, 2 are more lipophilic, which may result in a certain change in the nature of the distribution of these substances in plants and, consequently, a changed activity profile. However, it was expected that the lipophilic aromatic group in compounds 1 and 2 could be easily removed in a different cell location in plant tissues as a result of hydrolysis. Structures of all synthesized compounds were confirmed by 1 H and 13 C NMR, mass-spectrometry and elemental analysis data. 1 Н and 13 С NMR-spectra were recorded with «Bruker DRX-400» spectrometer operating at 400.13 MHz frequency, using DMSO-d 6 as solvent and TMS as an internal standard. Chemical shifts were measured with 0.01 ppm accuracy; coupling constants are reported in Hertz. Mass-spectrawere recorded on an inductively coupled plasma mass spectrometer XSeries II ICP-MS (Thermo Scientific Inc., USA). The melting points were determined using the melting point (temperature) apparatus Stuart SMP20 (UK).
O-i-Propyl-N-(2-hydroxyethyl)oxamate (4). 9.74 g (67 mM) ofdiisopropyloxalate in ethanol (3.4 ml) was placed in a round bottom flask equipped with a dropping funnel and magnetic stirrer. The solution was cooled on an ice water bath up to 0 °C. Then solution of 0.811 g (13.3 mmol) of the monoethanolamine in ethanol (7 ml) was added dropwise to a stirred cool mixture. After the addition the mixture was left to warm up to room tempеrature, filtered and concentrated under vacuum. The product obtained was pure enoughfor further transformations, yield 90% Compounds 3 and 4 are highly soluble in water.

Treatment and Experimental Design
Wheat seeds (Triticum aestivum L.) were obtained from Biosphere LLC, Fedorovka village, Staroshaigovsky District, Mordovia, Russia. These seeds were in the registry of the Customs Union. The seeds are certified according to the safety requirements specified in the technical regulations of the Customs Union. In accordance with the Declaration EAEC NRUD-RU.AB97.V.0093/19, the seeds are named: "selected wheat for germination".
Seeds that were uniform in size and plump without damaging the embryo were carefully selected, disinfected for 10 min with 0.2% sodium hypochlorite solution to prevent microbial and fungal infections, and then washed three times with distilled water. Subsequently, the water on the surface of the wheat seeds was absorbed with filter paper, and then they were dried in an oven at 30 °C for 48 hours. The dried seeds were stored at 5 °C in a refrigerator.
For comparison, we used Chlormequat chloride (CCC), as a plant growth regulator recommended by BASF for the CIS countries, Asia and Africa. CCC is designed to increase the mass of the root system; increase the resistance of plants to stress; increase the survival rate of productive shoots during the period of their further discharge; prevent root lodging. However, this is a chlorine containing salt and so one of the goals of the work is to replace this compound. Other plant growth regulators (N-[2-(2-oxo-l-imidazolidinyl) ethyI]-N'-plienylurea-EDU, 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea-TDZ were used, but these studies were not included in the article, because these two compounds are not widely used in Russia.
Three independent series of experiments were conducted: two in Petri dishesand a "pot" experiment. All the experiments were conducted simultaneously in the same room on a Phyto-LED NLO-79-01-00unit, which provides 12-hour illumination of samples with an intensity of at least 250 lux (plus 12 hours of darkness) and Red 615/Blue 457 nm wavelengths at 65% relative humidity.

The First Series of Experiments in Petri Dishes
Dry wheat seeds were taken in the first series of experiments. For this, a round support of filter paper of the appropriate size was placed in the cups and 50 seeds were spread over its surface; the seeds were pretreated with pump irrigation with dilute aqueous solutions of compounds 1-4 in a ratio of 10 mg L -1 . After that 50 dry seeds were germinated in Petri dishes with a threefold repetition of the experiment. Pump spraying was carried out in an insulated box, the surface of which, at the end of the procedure, was disinfected with steam and wiped with paper napkins. After spraying, the seeds were covered with filter paper of the appropriate diameter and filled with 10 ml of distilled water. Then Petri dishes with lids were placed on the shelves of the germinating device at 20 °C for 7 days. The first control group of seeds was treated with pump spray with distilled water, the second, with CCC with a concentration of 10 mg L -1 . Petri dishes with seeds were aired daily for 25 minutes by removing the lids, and, if necessary, 5-10 ml of distilled water was added to prevent the seeds from drying out.
On the third day of germination, the lids were removed from the Petri dishes so as not to interfere with the growth of seedlings. On the 7th day, according to the Formula (1), seed germination was determined. To measure the length of the root (RL) and shoot (SL), 15 seedlings were randomly selected from each Petri dish. In addition, RWC, SVI (I) and SVI (II) were calculated using Equations (2), (3) and (4), respectively. The measurement results are presented in Table 1 and Figures 2-6.

The Second Series of Experiments in Petri Dishes
In the second series of experiments about 320 wheat seeds were steeped in distilled water for 18 hours. After this period, the seeds were distributed between six Petri dishes. As a criterion of germination, the root of 1 mm was registered. Pump irrigation was performed as described in 2.2.1. A total of six procedures were conducted, each one repeated four times, so totally 24 Petri dishes were included in this study. Germination rate, RWC, SVI (I) and SVI (II) were calculated in the same way as in 2.2.1. The measurement results are presented in Table 2 and Figures 2-6, which also demonstrate a significant effect of compounds 1-4 on the germination of presoaked wheat seeds and the length of 7-day old seedlings.
For this purpose, dry wheat seeds were placed in Petri dishes, 50 pieces per each. Spraying was performed as described in 2.2.1. Then the treated seeds were transferred into plastic pots with a pre-moistened soil, sprinkled with soil and left for germination on a phyto-LED lighting unit providing illumination of samples 12 hours a day at 20 °С. Seeds sprayed with distilled water and placed in plastic pots were chosen as the first control group. The second control group of seeds in a Petri dish was treated by spraying CCC with concentration of 10 mg L -1 . Then the seeds were also transferred into plastic pots. In total, six procedures were performed; each procedure was repeated three times. Two weeks after the start of the third series of experiments, the watering regime of the plants was suspended for seven days. The duration of this experiment was 30 days. Germination, RWC, SVI (I) and SVI (II) were calculated in the same way as in paragraph 2.2.1. The measurement results are shown in Tables 3-5.

Statistical Analysis
Statistical processing of the results was performed using Microsoft Excel software and STATISTICA 13.3 TRIAL (StatSoft Russia). Basic statistical parameters such as mean, standard deviation (SD) were computed along with one-way analysis of variance (ANOVA). To assess the statistical significance of various data sets, an acceptable value of significance was p ≤ 0.05. The 95% confidence interval of true averages is shown in the Tables 1-3.

Seed Germination
In the present study, we observed that for all the tested compounds 1-4 in the first and second series of experiments (Tables 1 and 2; Figure 2), there was a significant increase in germination results. The active ingredients softened the shell of wheat seeds, penetrated into the grain and activated physiological processes. This had a beneficial effect on their germination. Thus, the highest germination rate (89.6-96.3%) was registered jas.ccsenet.
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Seed vigou as "the sum seed or se seed vigou Using com presented values (I a the third s control gr photosynth Figure 5. ur is an import m total of tho eed lot during ur index is con mpounds 1, 3 in Tables 1-3  wing mental o the percentage of germination. When using compounds 1 and 2, some of the plants did not recover and died, although less than in the absence of any treatment. The best results were demonstrated for plants treated with substances CCC and 4 in the complete absence of withered plants, as well as for compound 3. The length of the root and shoots was determined at the time of the end of the experiment. Plants treated with compounds 1, 3 and 4 were 21% higher than those of the first control group and 17% higher than the plants of the second control group (Table 3). The relative water content (RWC) was 82±10% in all plants at the end of the third series of experiments. This indicates that all samples survived a water deficit.

Conclusion
The data obtained as a result of biological tests indicate that the synthesized compounds 1-4 with functional groups separated by ethylene spacer are characterized by the expressed antistress and growth regulatory activity. The active substances softened the shell of wheat seeds, penetrated into the grain and activated physiological processes, which subsequently had a beneficial effect on their germination. Their use result in a significant increase of germination, an increase in the index of plant viability, it has a stimulating effect on the growth of shoots and roots in this wheat variety, as well as it improves resistance to water stress and the formation of dry matter. Plants treated with compounds 3 and 4 further improve their ability to fully regenerate after lodging as a result of a weekly drought. Thus, the obtained new synthetic antistress drugs increase the resistance of wheat plants (Triticum aestivum L.) to negative environmental factors and exhibit growth regulating activity.