Acta agriculturae Slovenica, 118/4, 1–7, Ljubljana 2022 doi:10.14720/aas.2022.118.4.2438 Original research article / izvirni znanstveni članek Antioxidant response of Impatiens walleriana L. to drought Anamarija MATIJEVIĆ 1, Ajla ŠAKONJIĆ 1, Senad MURTIĆ 1, 2 Received November 25, 2021; accepted October 17, 2022. Delo je prispelo 25. novembra 2021, sprejeto 17. oktobra 2022 1 University of Sarajevo, Faculty of Agriculture and Food Sciences, Department of Plant Physiology, Sarajevo, Bosnia and Herzegovina 2 Corresponding author, e-mail: murticsenad@hotmail.com Antioxidant response of Impatiens walleriana L. to drought Abstract: Stress caused by drought induces plant mor- phology, biochemistry, and physiology changes, leading to considerable reductions in plant growth and productivity. This study aimed to evaluate the antioxidant defence system of im- patiens seedlings (Impatiens walleriana L.) under drought. The antioxidant response of impatiens to drought was evaluated us- ing following parameters: the activity of catalase, guaiacol per- oxidase, pyrogallol peroxidase and ascorbate peroxidase, total phenolic and flavonoids contents and total antioxidant capacity. The experiment was conducted during 2020 in a greenhouse under controlled conditions. Half of the impatiens seedlings (20 plants), after the acclimation period in the greenhouse, were exposed to drought for a period of five days, while the sec- ond half was not (20 plants were regularly watered). The results of the study showed that the exposure of impatiens seedlings to drought increased the activity of enzymatic components, total phenolics and flavonoids contents and total antioxidant capacity of leaves. Greater exposure of impatiens to drought (in the observed period) implied a higher plant enzymatic and non-enzymatic antioxidant defence system activity. These re- sults confirm that impatiens has evolved both enzymatic and non-enzymatic antioxidant defence mechanisms to adapt and survive the short-term drought exposure. Key words: defence system; free radicals; leaves; plant growth; stress Antioksidacijski odziv vodenke (Impatiens walleriana L.) na sušo Izvleček: Stres, ki ga povzroča suša sproži v rastlinah spre- membe v morfologiji, biokemični zgradbi in fiziologiji, kar vodi k znatnemu zmanjšanju rasti in produktivnosti rastlin. Namen raziskave je bil ovrednotiti antioksidacijsko obrambo sejank vodenke (Impatiens walleriana L.) v sušnem stresu. Antioksi- dacijski odziv vodenke na sušo je bil ovrednoten z naslednjimi parametri: aktivnostjo katalaze, guajakol peroksidaze, pirogalol peroksidaze in askorbat peroksidaze, vsebnostjo celokupnih fe- nolov in flavonoidov in celokupne antioksidacijske kapacitete. Poskus je bil izveden v rastni sezoni 2020 v rastlinjaku v nadzo- rovanih razmerah. Polovica sejank vodenke (20 rastlin), je bila po aklimatizaciji razmeram rastlinjaka izpostavljena sušnemu stresu za pet dni, medtem ko je bila druga polovica (20 rastlin) redno zalivana. Rezultati raziskave so pokazali, da je izpostavi- tev sejank vodenke sušnemu stresu povečala aktivnosti analizi- ranih encimov, vsebnosti celokupnih fenolov in flavonoidov ter celokupno antioksidacijsko sposobnost listov. Večja izpostavi- tev vodenk suši je v opazovanem obdobju povzročila večji en- cimski in neencimski antioksidacijski obrambni odziv. Rezulta- ti potrjujejo, da ima vodenka sposobnost razvoja encimskega in neencimskega antioksidacijskega obrambnega sistema in lahko preživi krajša obdobja izpostavitve suši. Ključne besede: obrambni sistem; prosti radikali; listi; rast rastlin; stres Acta agriculturae Slovenica, 118/4 – 20222 A. MATIJEVIĆ et al. 1 INTRODUCTION Drought is the most important abiotic factor limit- ing crop productivity. The lack of water in soil reduces the soil water potential and the ability of plants to take up water, resulting in growth inhibition and reproduc- tive failure (Fahad et al., 2017). In addition, the inevitable consequence of drought is an increase in the production of reactive oxygen species (ROS) in plant cells. ROS in- clude free radicals such as superoxide radical, hydroxyl radical as well as non-radical molecules like hydrogen peroxide (H2O2). Increased levels of ROS can cause cel- lular damage and even cell death (Tola et al., 2021). Plants, however, have evolved numerous mecha- nisms to contend with oxidative stress, including the enzymatic and non-enzymatic antioxidant systems. Non-enzymatic defences include compounds with anti- oxidant properties such as phenolic compounds, vitamin C and carotenoids, while the enzymatic defences include antioxidant enzymes associated with ROS scavenging in plants such as superoxide dismutase (SOD), guaiacol peroxidase (GPX), pyrogallol peroxidase (PPX), ascor- bate peroxidase (APX) and catalase (CAT) (Mehla et al., 2017). SOD protects cells against ROS by catalysing the dismutation of highly toxic superoxide anions to less tox- ic hydrogen peroxide (H2O2) and molecular oxygen (O2). After dismutation of the superoxide anions by SOD into O2 and H2O2, the CAT decomposes the released H2O2 into H2O and O2 (Berwal & Ram, 2018). GPX and PPX also protect cells against the damaging effect of H2O2 by catalysing their decomposition through oxidation of phenolic substrates (Gill & Tuteja, 2010). APX is also a H2O2-scavenging enzyme. APX utilizes ascorbic acid as specific electron donor to reduce H2O2 to H2O (Hasanuz- zaman et al., 2019). The aim of this study was to evaluate the enzymatic and non-enzymatic antioxidant defence system of impa- tiens seedlings (Impatiens walleriana L.) under drought stress. Impatiens was selected as subject of this study primarily because the global production of this flower- ing plant species is consistently increasing. Therefore any new knowledge about the behaviour of these plants, especially under stress conditions, is of great interest to both producers and scientists. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL CONDITIONS The experiment was conducted in May 2020 under controlled conditions in the greenhouse of public com- munal company ’Park’ in Sarajevo. The temperature in the greenhouse during the experiment was maintained at 24 °C/21 °C during day/night, while the relative humidity (RH) was maintained between 60 % and 70 %, with com- bined venting to reduce RH and fogging systems to in- crease RH. In the beginning of the experiment, the impatiens seedlings were in the initial stage of flowering. The first part of the study involved transplanting impatiens seedlings into individual pots (20 cm diameter × 13 cm height), containing substrate Florahum-SP. Impatiens seedlings used in the experiment were produced in the nursery near the greenhouse and showed no significant difference in size and appearance. Ten days after transplanting, half of the impatiens (20 plants) were exposed to drought for next five days (non-watering). However, the second half was not ex- posed to drought, they served as controls (20 plants were regularly watered). Leaves of impatiens were sampled at the beginning and at the end of experiment (2nd and 5th day after drought treatment). Each leaf sample consisted of three fully expanded and healthy impatiens leaves. Fresh leaves were cut and immediately frozen with liquid nitrogen and then stored in ultra-freezer at -20 °C until further use. 2.2 PROTEIN AND ENZYME ACTIVITY MEAS- UREMENTS To obtain the extracts that were used to determine the protein content and activities of catalase and peroxi- dases, 0.5 g of fresh leaf sample was macerated using a mortar and pestle with liquid nitrogen and 0.015 g poly- vinylpyrrolidone (PVP). The powder thus obtained was homogenized in 1.5 ml 50 mM potassium phosphate buffer (pH 7) containing 1 mM dithiothreitol (DTT) and 1mM ethylenediaminetetraacetic acid (EDTA). The ho- mogenized material was centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant was used for protein and enzyme activity measurements. The extracted proteins were quantified using the Bradford method with bovine serum albumin (BSA) as the standard (Bradford, 1976). The pyrogallol peroxidase (PPX) activity was determined by the oxidation of py- rogallol according to the method of Chance & Maehly (1955) and the results were expressed as µmol purpuro- gallin per min per mg protein. The guaiacol peroxidase (GPX) activity was determined by the oxidation of guai- acol according to the method of Chance & Maehly (1955) and the results were expressed as µmol tetraguaiacol per min per mg protein. The ascorbate peroxidase (APX) ac- tivity was determined by the oxidation of ascorbic acid Acta agriculturae Slovenica, 118/4 – 2022 3 Antioxidant response of Impatiens walleriana L. to drought according to the method of Nakano & Asada (1981) and the results were expressed as µmol ascorbic acid oxidized per min per mg protein. The catalase (CAT) activity was determined by monitoring the decrease in absorbance at 240 nm at an interval of 5 to 120 sec as a result of H2O2 consumption (Aebi, 1984). Results were expressed as µmol of H2O2 consumed per min per mg protein. 2.3 EXTRACTION OF PHENOLIC COMPOUNDS FROM LEAVES The collected fresh leaves of impatiens were oven- dried at 40  °C (3 days) to avoid degradation of their phenolic compounds. After that, dried leaf samples were ground to a fine powder using an electric blender and stored at 4 °C until extraction and analysis. Extraction of phenolic compounds from dried leaf sample was done as follows: 1 g of sample was extracted with 30 ml of 60 % ethanol aqueous solution at room temperature for 24 h. Thereafter, the extract was filtered through Whatman fil- ter paper (11 μm pore size) into 50 ml volumetric flask and diluted to the mark with 60 % ethanol aqueous solu- tion. The extract thus obtained was used to estimate total phenolic content, total flavonoid content and total anti- oxidant capacity. 2.4 TOTAL PHENOLICS CONTENT The colorimetric reaction with Folin-Ciocâlteu rea- gent was performed to determine the content of phenolic compounds in leaf samples of impatiens (Ough & Amer- ine, 1988). The reaction mixtures consisted of 0.1 ml of extract, 6 ml of distilled water, 0.5 ml of Folin-Ciocalteu reagent (before use diluted in distilled water 1:2, v/v) and 1.5 ml of 20 % Na2CO3 were mixed thoroughly. Thereaf- ter, the mixture was heated in a water bath at 40 oC for 30 min. After cooling to room temperature, the absorbance of the mixture was read at 765 nm. The results were cal- culated on the basis of the calibration curve for gallic acid (0-500 mg l-1) and were expressed as mg of gallic acid equivalents per g of dry mass (mg GAE g-1 DM). 2.5 TOTAL FLAVONOIDS CONTENT The aluminum chloride colorimetric assay was performed to determine the total flavonoid contents (Zhishen et al., 1999). The reaction mixtures consisted of 1 ml of extract, 4 ml of distilled water, 0.3 ml of 5 % NaNO2, 0.3 ml of 10 % AlCl3 and 2 ml of 1 M NaOH were mixed thoroughly. The mixture was made up to 10 ml with distilled water and incubated at room temperature for 1 h, and then the absorbance of the mixture was read at 510 nm. The results were calculated on the basis of the calibration curve for catechin (0-100 mg l-1) and were ex- pressed as mg of catechin equivalents per g of dry mass (mg C g-1 DM). 2.6 FERRIC REDUCING ANTIOXIDANT POWER (FRAP) ASSAY Ferric reducing antioxidant power (FRAP) assay was performed to estimate the total antioxidant capacity (Benzie & Strain, 1996). The reaction mixture consisted of 80 μl of extract, 240 μl of distilled and 2080 μl of fresh FRAP reagent were mixed thoroughly. The FRAP reagent was prepared immediately before use by mix- ing acetate buffer (300 mM, pH = 3.6), 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM HCl and 20 mM FeCl3 in a volume ratio of 10:1:1. Thereafter, the mixture was heated at 37 °C for 15 min in a water bath. After cooling to room temperature the absorbance was read at 595 nm. The results were calculated on the basis of the calibration curve for FeSO4 × 7 H2O (0-2000 µM) and were expressed as μmol of Fe2+ per g of dry mass (μmol Fe2+ g-1 DM). Amersham ultrospec 2100 spectro- photometer (Biochrom, USA) was used for all spectro- photometric measures. 2.7 STATISTICAL ANALYSIS All experimental measurements were done in trip- licates and the results were presented as mean ± stand- ard deviation. Pearson correlation coefficient was used to reflect relationship total phenolic, total flavonoids and total antioxidant activities. One-way analysis of variance (ANOVA) and least-significant-difference test (LSD) at 0.05 level of probability (p < 0.05) were performed to evaluate statistical significance between the means using Microsoft Excel 2010 software (Office 2010, Redmond, WA, USA). 3 RESULTS The activity of all tested enzymes (GPX, PPX, APX and CAT) in the observed period were higher in leaves of impatiens exposed to drought compared to impa- tiens grown under standard growth conditions (without stress), as shown in Table 1. The results of this study also showed that the activities of all enzymes were increased with the progress of stress. Acta agriculturae Slovenica, 118/4 – 20224 A. MATIJEVIĆ et al. The activity of GPX and APX were significantly higher in the leaves of impatiens exposed to drought than in the control, regardless of the duration of plant exposure to stress. However, the activity of PPX and CAT in leaves of all stressed impatiens seedlings was signifi- cantly higher only at the end of the experiment i.e. on the fifth day of plant exposure to drought. In controls i.e. in variants where impatiens seedlings were not exposed to drought, the activity of all enzymes tested did not change significantly during the experiment. In this study, the non-enzymatic antioxidant de- fence system (total phenolic contents (TPC), total flavo- noids (TFC) and total antioxidant capacity (TAC) were also affected by growth conditions (Table 2). As shown in Table 2, TPC, TFC and TAC were high- er in the leaves of impatiens exposed to drought than in control. The increases were statistically significant for both the second and the fifth day of plant exposure to drought. In this study, there was a positive and strong signifi- cant relationship between the total phenolic/flavonoids and the total antioxidant capacity of impatiens leaves re- gardless of growth conditions, indicating that phenolic compounds are mainly responsible for total antioxidant capacity of plants (Table 3). 4 DISCUSSION A key sign of drought stress at the cellular level is the overproduction of reactive oxygen species (ROS), which is being considered as the most common cause of cellular damage. However, plants have evolved an efficient enzy- matic and non-enzymatic antioxidant system to protect themselves against ROS. Within a cell, the SOD consti- tutes the first line of plant antioxidant defence against ROS. However, H2O2, which results from the action of SOD, is toxic to cells. Therefore, the efficient scavenging of H2O2 is regarded as a key feature in the cellular antioxi- dant defence system. Fortunately, plant cells are endowed with H2O2-metabolizing enzymes such as peroxidases and catalase. Peroxidases are group of enzymes that cata- lyse the conversion H2O2 into H2O using a wide variety of substrates as an electron donor (Abedi & Paknyat, 2010). In this study, generally, stress caused by drought increased the CAT and peroxidase enzymatic activity, and the increase was in line with stress duration; greater exposure of impatiens to drought (in the observed pe- riod) implied a higher activity of antioxidant enzymes. However, there was a differential level of activity among enzymes. The activities of GPX and APX enzymes at the early stage of drought stress (2nd day after drought treat- ment) were significantly higher as compared to CAT, although both peroxidases and CAT act on the same substrate (H2O2). Lower CAT activities in plants at the early stage of stress have been reported in many stud- ies (Chugh et al, 2013; Antonić et al., 2016; Wang et al., 2019). Smirnof & Araound (2019) noted that CAT does not have a high affinity for H2O2 and this is probably one of the main reasons for its low activity. However, CAT has Treatment Enzyme activity (µmol min-1 mg-1 protein) GPX PPX APX CAT 2nd day of exposure to drought 0.25 ± 0.11b 0.41 ± 0.13b 0.27 ± 0.26b 0.009 ± 0.001b 2nd day without stress 0.16 ± 0.07c 0.33 ± 0.21b 0.13 ± 0.12c 0.008 ± 0.002b 5th day of exposure to drought 0.31 ± 0.04a 1.03 ± 0.13a 0.51 ± 0.16a 0.033 ± 0.014a 5th day without stress 0.18 ± 0.04c 0.48 ± 0.07b 0.22 ± 0.08bc 0.009 ± 0.004b LSD0.05 0.054 0.159 0.114 0.006 Table 1: Effect of short-term exposure to drought on antioxidant enzymes of impatiens leaves Treatment TPC (mg GAE g-1 DM) TFC (mg C g-1 DM) TAC (μmol Fe2+ g-1 DM) 2nd day of exposure to drought 6.92 ± 0.18b 2.08 ± 0.24b 92.91 ± 5.82b 2nd day without stress 5.65 ± 0.22c 1.50 ± 0.18c 65.23 ± 3.65c 5th day of exposure to drought 7.98 ± 0.70a 2.68 ± 0.34a 103.95 ± 4.17a 5th day without stress 6.37 ± 0.75bc 2.20 ± 0.20b 89.42 ± 12.38b LSD0.05 0.83 0.26 10.58 Table 2: Effect of short-term exposure to drought on non-enzymatic antioxidants of impatiens leaves Acta agriculturae Slovenica, 118/4 – 2022 5 Antioxidant response of Impatiens walleriana L. to drought ity of antioxidant defence mechanisms depends on each phenolic compound’s chemical structure. Among the phenolic compounds with known antioxidant activ- ity, flavonoids are highlighted (Dibacto et al., 2021). In this study, TFC in leaves of impatiens were progressively influenced by drought. An increase in TFC in leaves of impatiens was already recorded in the 2nd days after drought treatment, and with the progress of stress (5th days after drought treatment), TFC was gradually in- creased. In this study, an increase of TFC was in line with increase of TPC in impatiens leaves regardless of growth conditions. This was expected since the flavonoids are the biggest group of phenolic compounds. In the present study, the total antioxidant capacity level estimated with FRAP assay was also significantly higher in leaves of impatiens exposed to drought than in controls. Furthermore, the present study indicates a very strong relationship between the TPC/TFC and TAC in leaves of impatiens, regardless of growth conditions. In short, the antioxidant activity in leaves of impatiens increased by increasing the total phenolic and flavo- noid contents. These results were also expected since it is known that phenolic compounds are among the most potent antioxidants from plants. The levels of enzymatic and non-enzymatic antioxi- dants in impatiens leaves were very high in the fifth day after drought treatment. Accumulation of these antioxi- dants suggests a high level of stress convened to the impa- tiens during this period (Sharma et al., 2012). It can also be assumed that the impatiens during this period con- tinues to defend itself against ROS by producing a high amount of enzymatic and non-enzymatic antioxidants (Kim et al., 2014). However, numerous studies reported a decline in the activity of antioxidant enzymes in various plants in the final stage of stress (three days or more after exposed plant to stress), indicating that antioxidant ca- pacity and thus drought tolerance can vary among plants (Almeselmani et al., 2006; Sabzmeydani et al., 2021). It is evident that plant response to drought depends not only on the extremity and time duration of the stress but also on the plant genetic background. 5 CONCLUSIONS Exposure of impatiens seedlings to drought in- creased the activity of enzymatic antioxidants, total phe- nolic and flavonoid contents and total antioxidant capac- ity of leaves. Greater exposure of impatiens to drought (in the observed period) implied a higher activity of plant enzymatic and non-enzymatic antioxidant de- fence systems. These results confirm that impatiens have evolved both enzymatic and non-enzymatic antioxidant a very high reaction rate (Smejkal & Kakumanu, 2019). The Braunschweig Enzyme Database (BRENDA) reports that one molecule of catalase can convert over 2.8 million molecules of hydrogen peroxide to water and oxygen per second (Schomburg et al., 2017). Therefore, CAT is a sink for H2O2 and is indispensable for plant defence system against oxidative stress (Willekens et al., 1997). Besides enzymatic antioxidants, plants synthesize a wide range of non-enzymatic antioxidants capable of decreasing ROS-induced oxidative damage (Kasote et al., 2015). Non-enzymatic antioxidants include vitamin C, vitamin E, phenolic compounds, carotenoids, etc. Among all non-enzymatic antioxidants, phenolic com- pounds appear to be the most important since they have a great potential to clear ROS. The antioxidant proper- ties of phenolic compounds are mainly due to their high redox potential, allowing them to act as reducing agents, hydrogen donors or singlet oxygen quenchers (Liang et al., 2010). In the present study, the accumulation of phenolic compounds was significantly higher in leaves of im- patiens exposed to drought than in controls (without stress). Moreover, an increase in phenolics contents was more significant in impatiens exposed to drought for longer duration. These results suggest that plant initi- ates the intensive synthesis of phenolic compounds as a response to drought, and this hypothesis has been con- firmed by many other scientists (Basu et al., 2010; Cram- er et al., 2011; Šamec et al., 2021). Sharma et al. (2019) reported that the considerable accumulation of phenolic compounds in plants is usu- ally a consistent feature of non-enzymatic antioxidant defence mechanisms under stress. However, the capac- Treatment TPC TFC TAC 2nd day of exposure to drought TPC 1 0.95 0.93 TFC 1 0.94 TAC 1 2nd day without stress TPC 1 0.92 0.93 TFC 1 0.91 TAC 1 5th day of exposure to drought TPC 1 0.94 0.95 TFC 1 0.96 TAC 1 5th day without stress TPC 1 0.93 0.94 TFC 1 0.92 TAC 1 Table 3: Pearson’s correlation between total phenolic (TPC), total flavonoids (TFC) and total antioxidant capacity (TAC) Acta agriculturae Slovenica, 118/4 – 20226 A. MATIJEVIĆ et al. defence mechanisms to adapt and survive the short-term drought exposure. 6 REFERENCES Abedi, T., & Pakniyat, H. (2010). Antioxidant enzyme changes in response to drought stress in ten cultivars of oilseed rape (Brassica napus L.). Czech Journal of Genetics and Plant Breeding, 46, 27–34. https://doi.org/10.17221/67/2009- CJGPB Aebi, M. (1984). Catalase in vitro. Methods in Enzymology, 105, 121–126. https://doi.org/10.1016/s0076-6879(84)05016-3 Almeselmani, M., Deshmukh, P.S., Sairam, R.K., Kushwaha, S.R., Singh, T.P. (2006). Protective role of antioxidant en- zymes under high temperature stress. Plant Science, 171(3), 382–388. https://doi.org/10.1016/j.plantsci.2006.04.009 Antonić, D., Milošević, S., Cingel, A., Lojić, M., Trifunović- Momčilov, M., Petrić, M., Subotić, A., Simonović, A. (2016). Effects of exogenous salicylic acid on Impatiens walleri- ana L. grown in vitro under polyethylene glycol-imposed drought. South African Journal of Botany, 105, 226–233. https://doi.org/10.1016/j.sajb.2016.04.002 Basu, S., Roychoudhury, A., Saha, P.P, Sengupta D.N. (2010). Differential antioxidative responses of indica rice cultivars to drought stress. Plant Growth Regulation, 60(1), 51–59. https://doi.org/10.1007/s10725-009-9418-4 Benzie, I.F., & Strain, J.J. (1996). Ferric reducing ability of plas- ma (FRAP) as a measure of antioxidant power: The FRAP assay. Analytical Biochemistry, 239(1), 70–76. https://doi. org/10.1006/abio.1996.0292 Berwal, M.K. & Ram, C. (2018). Superoxide Dismutase: A Stable Biochemical Marker for Abiotic Stress Tolerance in Higher Plants. In A. B. De Oliveira (Ed), Abiotic and Bi- otic Stress in Plants. London, UK: IntechOpen. https://doi. org/10.5772/intechopen.82079 Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochem- istry, 72, 248–254. https://doi.org/10.1006/abio.1976.9999 Chance, B., & Maehly A.C. (1955). Assay of catalases and per- oxidases. Methods in Enzymology, 2, 764–775. https://doi. org/10.1016/S0076-6879(55)02300-8 Chugh, V, Kaur, N, Grewal, M.S., Gupta, A.K. (2013). Differen- tial antioxidative response of tolerant and sensitive maize (Zea mays L.) genotypes to drought stress at reproductive stage. Indian Journal of Biochemistry and Biophysics, 50(2), 150–158. Cramer, G.R., Urano, K., Delrot, S., Pezzotti, M., Shinozaki, K. (2011). Effects of abiotic stress on plants: a systems bio- logy perspective. BMC Plant Biology, 11, 163. https://doi. org/10.1186/1471-2229-11-163 Dibacto, R.E.K., Tchuente,B.R.T., Nguedjo, M.W., Tientch- eu, Y.M.T., Nyobe, E. C., Edoun, F.L.E., Kamini, M.F.G., Dibanda, R.F., Medoua, G.N. (2021): Total polyphenol and flavonoid content and antioxidant capacity of some varieties of Persea americana peels consumed in Cam- eroon. Scientific World Journal, 2021, e8882594. https://doi. org/10.1155/2021/8882594 Fahad, S., Bajwa. A.A., Nazir, U., Anjum, S.A., Farooq, A., Zo- haib, A., Sadia, S., Nasim, W., Adkins, S., Saud, S., Ihsan, M.Z., Alharby, H., Wu, C., Wang, D., Huang, J. (2017). Crop production under drought and heat stress: Plant respons- es and management options. Frontiers in Plant Science, 8, e1147. https://doi.org/10.3389/fpls.2017.01147 Gill, S.S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48(12), 909–930. https://doi.org/10.1016/j.plaphy.2010.08.016 Hasanuzzaman, M., Bhuyan, M., Anee, T.I., Parvin, K., Nahar, K., Mahmud, J.A., Fujita, M. (2019). Regulation of ascor- bate-glutathione pathway in mitigating oxidative damage in plants under abiotic stress. Antioxidants, 8(9), 384. https://doi.org/10.3390/antiox8090384 Kasote, D.M., Katyare, S.S., Hegde, M.V., Bae, H. (2015). Signif- icance of antioxidant potential of plants and its relevance to therapeutic applications. International Journal of Biological Sciences, 11(8), 982–991. https://doi.org/10.7150/ijbs.12096 Kim, Y.H., Khan, A.L., Kim, D.H., Lee, S.Y., Kim, K.M., Waqas, M., Jung, H.Y., Shin, J.H., Kim, J.G., Lee, I.J. (2014). Sili- con mitigates heavy metal stress by regulating P-type heavy metal ATPases, Oryza sativa low silicon genes, and endog- enous phytohormones. BMC Plant Biology, 14, 13. https:// doi.org/10.1186/1471-2229-14-13 Liang, T., Yue, W., Li, Q. (2010): Comparison of the phenolic content and antioxidant activities of Apocynum venetum L. (Luo-Bu-Ma) and two of its alternative species. Internation- al Journal of Molecular Sciences, 11(11):4452–4464. https:// doi.org/10.3390/ijms11114452 Mehla, N., Sindhi, V., Josula, D., Bisht, P., Wani, S.H. (2017). An introduction to antioxidants and their roles in plant stress tolerance. In M. I. R. Khan & N. A. Khan (Eds.), Reactive Oxygen Species and Antioxidant Systems in Plants: Role and Regulation under Abiotic Stress (pp. 1–23). Singapure, SG: Springer. https://doi.org/10.1007/978-981-10-5254-5_1 Nakano, Y., & Asada K. (1981). Hydrogen peroxide is scav- enged by ascorbate specific peroxidase in spinach chloro- plasts. Plant Cell Physiology, 22(5), 867–880. https://doi. org/10.1093/oxfordjournals.pcp.a076232 Ough, C.S., & Amerine, M.A. (1988). Methods for Analysis of Musts and Wines (pp. 196–221). New York, NY: John Wiley & Sons. Sabzmeydani, E., Sedaghathoor, S., Hashemabadi, D. (2021). Effect of salicylic acid and progesterone on physiological characteristics of Kentucky bluegrass under salinity stress. Revista de Ciencias Agrícolas, 38(1), 111–124. https://doi. org/10.22267/rcia.213801.151 Šamec, D., Karalija, E., Šola, I., Vujčić Bok, V., Salopek-Sondi, B. (2021). The role of polyphenols in abiotic stress response: The influence of molecular structure. Plants 10(1), 18. htt- ps://doi.org/10.3390/plants10010118 Schomburg, I, Jeske, L, Ulbrich, M, Placzek, S., Chang, A., Schomburg, D. (2017). The BRENDA enzyme informa- Acta agriculturae Slovenica, 118/4 – 2022 7 Antioxidant response of Impatiens walleriana L. to drought tion system–from a database to an expert system. Journal of Biotechnology 261, 194–206. https://doi.org/10.1016/j. jbiotec.2017.04.020 Sharma, A., Shahzad, B., Rehman, A., Bhardwaj, R., Landi, M., Zheng, B. (2019). Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules (Basel, Switzerland), 24(13), 2452. https://doi. org/10.3390/molecules24132452 Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M. (2012). Re- active oxygen species, oxidative damage, and antioxida- tive defense mechanism in plants under stressful con- ditions. Journal of Botany, 2012, e217037. https://doi. org/10.1155/2012/217037 Smejkal G.B., & Kakumanu S. (2019). Enzymes and their turn- over numbers. Expert Review of Proteomics, 16(7), 543–544. https://doi.org/10.1080/14789450.2019.1630275 Smirnoff, N., & Arnaud, D. (2019). Hydrogen peroxide me- tabolism and functions in plants. New Phytologist, 221(3), 1197–1214. https://doi.org/10.1111/nph.15488 Tola, A.J., Jaballi, A., Missihoun, T.D. (2021). Protein carbon- ylation: Emerging roles in plant redox biology and future prospects. Plants, 10(7), e1451. https://doi.org/10.3390/ plants10071451 Wang, X, Liu, H, Yu, F, Hu, B, Jia, Y, Sha, H, Zhao, H. (2019). Differential activity of the antioxidant defence system and alterations in the accumulation of osmolyte and reactive oxygen species under drought stress and recovery in rice (Oryza sativa L.) tillering. Scientific Reports 9(1), 8543. htt- ps://doi.org/10.1038/s41598-019-44958-x Willekens, H., Chamnongpol, S., Davey, M., Schraudner, M., Langebartels, C., Van Montagu, M., Inzé, D., Van Camp, W. (1997). Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. The EMBO journal, 16(16), 4806–4816. https://doi.org/10.1093/emboj/16.16.4806 Zhishen, J., Mengcheng, T., Jianming, W. (1999). The determi- nation of flavonoid contents in mulberry and their scaven- ging effects on superoxide radicals. Food Chemistry, 64(4), 555–559. https://doi.org/10.1016/S0308-8146(98)00102-2