Acta agriculturae Slovenica, 120/3, 1–10, Ljubljana 2024 doi:10.14720/aas.2024.120.3.18268 Original research article / izvirni znanstveni članek Biochemical and molecular characterization of bread wheat genotypes under drought stress: Implications for antioxidant defense mechanisms and genomic analysis Numan ECZACIOGLU 1, Begum Terzi AKSOY 1, Ozlem Ates SONMEZOGLU 1, Yakup ULUSU 1, 2 Received February 22, 2024; accepted June 12. 2024. Delo je prispelo 22. februar 2024, sprejeto 12. junij 2024 1 Faculty of Engineering, Department of Bioengineering, Karamanoglu Mehmetbey University, Karaman, Turkey 2 Corresponding author, e-mail: yakupulusu@kmu.edu.tr Biochemical and molecular characterization of bread wheat genotypes under drought stress: Implications for antioxidant defense mechanisms and genomic analysis Abstract: Plants face abiotic stresses like drought, salin- ity, and high temperature, which adversely affect growth and induce physiological and metabolic changes. Drought is a com- plex stress controlled by many genes, requiring investigation through molecular markers and biochemical characterization in wheat genotypes. This study involved eight bread wheat cultivars and two controls: drought-tolerant ‘Gerek-79’ and drought-sensitive ‘Sultan-95’. These were grown for 40 days and then subjected to 10 days of drought stress. Antioxidants and antioxidant enzyme activities, which neutralize ROS, are key resistance mechanisms against oxidative stress. Levels of polyphenol oxidase (PPO), peroxidase (POD), ascorbate per- oxidase (APX), and catalase (CAT) were measured. Plant re- sponses to stress included changes in photosynthetic pigments, total proteins, hydrogen peroxide, lipid peroxidation (MDA), and proline levels. POD showed the highest change in enzyme activity, while PPO was least affected. Chlorophyll b levels in- creased under stress across all varieties. Notably, proline levels, an abiotic stress marker, significantly rose by the 10th day of drought. Additionally, wheat genotypes were analyzed using drought-related SSR markers (Xwmc 89, Xwmc 118, Xwmc 304, Xgwm 337). This allowed evaluation of the impact of molecular characterization on biochemical changes under drought stress. Key words: bread wheat, drought stress, antioxidant en- zymes, proline, lipid peroxidation, hydrogen peroxide, photo- synthetic pigments Prepoznavanje biokemičnih in molekularnih označevalcev krušne pšenice v razmerah sušnega stresa, ki so vključeni v mehanizme antioksidacijske obrambe in genomske analize Izvleček: Rastline se izpostavljene abiotskim stresom kot so suša, slanost in visoke temperature, ki negativno vpli- vajo na rast in vzpodbudijo fiziološke in med njimi presnovne spremembe. Suša je kompleksni stres, ki ga uravnavajo številni geni, kar pri genotipih krušne pšenice zahteva preučevanje mo- lekularnih označevalcev in biokemičnih procesov. V to raziska- vo je bilo vključenih osem genotiopov krušne pšenice in dve kontroli: na sušo tolerantna sorta Gerek-79 in občutljiva sorta sorta Sultan-95. Vse rastline so bile gojene 40 dni v standardnih razmerah in nato izpostavljene sušnemu stresu za 10 dni. An- tioksidanti in antioksidacijski emcimi, ki nevtralizirajo aktivne zvrsti kisika (ROS) so ključni mehanizmi za odpravo oksida- cijskega stresa. Izmerjene so bile vsebnosti polifenol oksidase (PPO), peroksidaze (POD), askorbat peroksidaze (APX) in katalaze (CAT). Analiza odziva rastlin na stres je obsegala še meritve sprememb v vsebnosti fotosinteznih pigmentov, celo- kupnih beljakovin, vodikovega peroksida, spremembe v pero- ksidaciji maščob (MDA) in v vsebnosti prolina. POD je poka- zala največjo spremembo v aktivnosti med tem, ko je bila PPO najmanj prizadeta. Vsebnost klorofila b se je v stresnih razme- rah povečala pri vseh sortah. Značilno se je po desetih dneh suše povečala vsebnost prolina kot označevalca abiotičnega stresa. Dodatno so bili v genotipih krušne pšenice anlizirani na s sušo povezani SSR označevalci (Xwmc 89, Xwmc 118, Xwmc 304, Xgwm 337). To je omogočilo ovrednotenje biokemičnih sprememb v razmerah sušnega stresa na molekularni osnovi. Ključne besede: krušna pšenica, sušni stres, antioksidaci- jski encimi, prolin, peroksidacija lipidov, vodikov peroksid, fitosintezna barvila Acta agriculturae Slovenica, 120/3 – 20242 N. ECZACIOGLU et al. 1 INTRODUCTION Global crop production is threatened by the in- creased frequency of long-term water and heat stress caused by climate change (Arora, 2019). Drought caused by climate change is one of the important natural dis- asters that significantly impact the agricultural sector. Due to its geographical size and range, it has a complex structure and affects a large population (Javadinejad et al., 2021). Plants can face very different stress conditions throughout their lives. These stress factors can be biotic (pathogen, competition with other organisms, etc.) or abiotic (drought, salinity, radiation, chemical substances, high temperature or frost etc.). The ability of a plant to grow in a limited area of water is referred to as drought resistance (Kadam et al., 2012). Under drought stress, plants exhibit various metabolic changes such as protein synthesis, ROS accumulation, enzyme activity change, and pigment content (Chen et al., 2016). The response of plants to drought stress depends on plant growth (development), stress period and plant genetics (Beltrano & Ronco, 2008; Khan et al., 2012). Drought stress induces many common cellular reactions in plants. The stress leads to cellular dehydration, which causes osmotic stresses resulting in reduced cytosolic and vacuolar bulk (Wang et al., 2003). Except for ionic compounds, the early response of this plant to the plant is largely the same. The response of the plant to the stress condition is quite complex in terms of cellularity, and plants respond to stress by changing many biochemical and molecular mechanisms (genes, etc.) at tissue and plant levels (Bohnert & Jensen, 1996; Wang et al., 2003). As a result, there are numerous changes to the plant ad- aptation, such as reduction in growth, slowing of photo- synthesis, closing of stomata, transient increases in ABA levels, induction of gene expressions, accumulation of coherent soluble and protective proteins, increased levels of antioxidants and suppression of energy consumption pathways (Bartels & Sunkar, 2005). Proteins are essential components of living organ- ism. The most important indicator of wheat quality is the amount of protein and the quality of the existing protein (Finney et al., 1987). As a result of water loss, the interactions of hydro- phobic and hydrophilic amino acids in the structure of proteins with water are disrupted. So, proteins denatura- tion and enzymes inhibition occur. Proline is a molecular chaperone that protects proteins from degradation and regulates different enzyme activities. It has been sug- gested that proline accumulation in high plants is an an- tioxidant because it contributes to osmotic compliance and is effective in preserving the integrity of the plasma membrane by against to drought and salinity, metal tox- icity, oxidative stress and biotic stress factors (Mansour, 1998; ÖZDEMİR et al., 2012). Causing cross-linking and polymerization of membrane components, aldehydes inactivate receptors and membrane bound enzymes at Figure 1: Schematic diagram of the study Acta agriculturae Slovenica, 120/3 – 2024 3 Biochemical and molecular characterization of bread wheat genotypes under drought stress: Implications for antioxidant defense membrane. So, serious damage can also occur in mem- brane proteins. Malondialdehyde (MDA), a reactive al- dehyde, is formed as a result of the oxidation of lipids in the biological system. MDA inhibits the production of peroxidized unsaturated fatty acids in plant membranes, triggering transcriptional stress responses (Tenikecier, 2013). In biotic or abiotic stressed plants, the inactiva- tion of reactive oxygen species, which damage plant cells to vital components such as membrane lipids, proteins, enzymes, pigments and nucleic acids, is provided by the increase of antioxidant enzymes (Maheshwari & Dubey, 2009). ROS, such as hydrogen peroxide, superoxide an- ion, hydroxyl radical and singlet oxygen, are formed by partial reduction of molecular oxygen (Kireçci, 2012). ROSs cause degradation of membrane components, oxidation of protein sulfhydryl groups, formation of cy- toplasmic gel phase, and loss of membrane functions. Antioxidants protect cells from oxidative damage by cap- turing and detoxifying ROS. This system consists of anti- oxidants such as ascorbate, glutathione, α tocopherol and carotenoids with small molecular weight and enzymatic antioxidants such as SOD, CAT, ASPX and GR (Ahmad et al., 2008; Keleş & Öncel, 2002; Zhang Feng et al., 2004). Wheat is an important grain product that meets a large portion (22 %) of global food needs (Naderi et al., 2020). Due to population growth worldwide (Faostat, 2017), wheat production needs to be supported to meet food needs around the world. Determining the crop tol- erance mechanism in drought is important for wheat breeding programs (Gálvez et al., 2019). Landraces are more tolerant to stressful environments and temporary climatic conditions thanks to their agro-physiological characteristics and genetic structure (Mohammadi et al., 2015). For this reason, determining suitable wheat varieties against drought stress can contribute to creat- ing new varieties and benefit from creating new product plans with improved stress tolerance (Naderi et al., 2020; Zhou et al., 2015). In this study, ten different bread wheat genotypes were subjected drought stress by deficient of water for 10 days. Changes in some antioxidant enzymes and molecular indicator of drought stress such as MDA, prolin, pigments etc. were measured in different wheat genotypes to develop relationship between antioxi- dant enzymes, molecular indicators and mechanisms of drought tolerance. 2 MATERIAL & METHOD 2.1 PLANT MATERIALS Ten bread wheat cultivars (Sarıcanak-98, Karahan, Sonmez2001, Kate-1, Altay2000, Bayraktar, Harman- kaya99, Izgi2001, Gerek-79, Sultan-95) that differ in drought tolerance were used as plant material. Gerek 79 cultivars for drought tolerance and Sultan 95 cultivars for drought sensitivity were used as control cultivars. The wheat cultivars and lines were kindly provided by the GAP International Agricultural Research Institute, Eskişehir Transition Zone Agricultural Research Insti- tute, Bahri Dağdaş International Agricultural Research Institute, Ankara Field Crops Central Research Institute and Thrace Agricultural Research Institute. Surface sterilization was applied before the wheat seeds were planted in petri dishes. Briefly, seeds were treated for 1 min with 70 % ethanol and 5 % sodium hy- pochlorite for 10 min. Finally, the seeds were rinsed three times with distilled water. The seeds were placed in the petri dishes with 10-12 seeds each. The petri dishes were closed with para-film and were kept at +4 °C for 4-5 days. Then, the germi- nation was observed for 3-4 days at room temperature. Seven- and ten-day seedlings pots were grown in pots for thirty days. Irrigation was left in the application groups after the thirtieth day. On the third, seventh, and tenth days, tissue samples were collected and stored at -80 °C for analysis. 2.2 DETERMINATION OF ANTIOXIDANT EN- ZYME ACTIVITIES 0.25 g of plant tissue was homogenized with potas- sium phosphate (pH = 7) and centrifuged at 10000 x G for 15 minutes. The supernatant was used to determine enzyme activities. Depending on the consumption of the prepared substrates or the product formation, the enzyme activi- ties were examined at the optimum pH and temperature for the enzymes by spectrophotometric method at spe- cific wavelengths. All characterization operations were performed in triplicate. 2.2.1 CATALASE ASSIGNMENT In the activity measurement, 3 ml of reaction buffer was prepared which including 50 mM phosphate buffer (pH = 7), 30 mM H₂O₂ and 30 μl homogenate. The ab- sorbance was measured during 2 minutes. For the cal- culations, the amount of enzyme that cleaved 1 μmol of H₂O2 was determined in one minute at 240 nm from using the A = Є.b.c formula by taking the light path as 10 mm and the extinction coefficient (Є H2O2: 0.0394 mmol-1x mm-1). Acta agriculturae Slovenica, 120/3 – 20244 N. ECZACIOGLU et al. 2.2.2 PEROXIDASE ASSIGNMENT To determine the peroxidase activity 3 ml of the re- action mixture was prepared consisting of 50 mM phos- phate buffer (pH = 7), 22.5 mM H2O2, 30 mM guaiacol and 20 μl enzyme extract. The activity to the reaction was initiated by the addition of the enzyme solution to the assay medium and optical density was recorded at 470 nm for 2 minutes. An enzyme unit was calculated as the amount of enzyme catalyzing 1 μmol guaiacol per min- ute. 2.2.3 ASCORBATE PEROXIDASE ASSIGNMENT To determine the ascorbate peroxidase activity, 3 ml of the reaction mixture was prepared in the activity measurement to consist of 100 mM phosphate buffer (pH = 7), 10 mM H2O2, 2.5 mM ascorbic acid and 100 μl enzyme extract. The reaction was initiated by adding hydrogen peroxide to the activity measurement medium last and the absorbance was recorded at 290 nm for 2 minutes. Enzyme activity was calculated using ascorbate extinction coefficient (2.8 mM-1xcm-1). 2.2.4 POLYPHENOL OXIDASE ASSIGNMENT 0.3 g of the tissue was taken and 2 ml of buffer (0.2 M Na2HPO4 and 5 mM ascorbic acid pH: 6.5) was added to prepare the homogenate. Homogenate was centrifuged at 12000 x G for 10 minutes. Enzyme activity measurement is based on the observation of the increase in absorbance at 420 nm caused by the conversion of catechol to brown- ish-yellow colored quinone in the presence of oxygen. 2.2.5 THE HYDROGEN PEROXIDE (H2O2) AS- SIGNMENT Leaf tissues were homogenized in 5 ml of ice-bath with 0.1 % (w / v) TCA. The homogenate was centrifuged for 15 min at 12000 x G. 0.5 ml of supernatant were add- ed to 0.5 ml of 10 mM potassium phosphate (pH: 7.0) buffer and 1 ml of 1 M KI. The absorbance of the mixture was then determined at 390 nm and the amount of H2O2 was determined by using the standard curve. 2.3 PHOTOSYNTHETIC PIGMENTATION AS- SIGNMENT Chlorophyll a, b and carotenoid amounts were determined according to Arnon after extraction with 80 % acetone of plant leaves (Arnon, 1949). The amount of carotenoid was determined according to the Jaspars formula (Ulusu et al., 2017). 0.2 g of leaf tissue was ho- mogenized in 5 ml porcelain mortar with 80 % acetone. The homogenate was transferred to a 15 ml centrifuge tube and centrifuged at 3000 x G for 5 minutes. The ab- sorbance of the supernatant at 645, 663 and 450 nm was measured by spectrophotometer and the amount of pig- ment/ml extract was calculated from the following for- mulas with the help of these absorbance values. Chlorophyll a = 12.7 x A663 – 2.69 x A645 Chlorophyll b = 22.9 x A645 – 4.68 x A663 Total Chlorophyll = 20.2 x A645 + 8.02 x A663 Carotenoid = 4.07 x A450 – (0.0435 x amount of Chlorophyll a + 0.3367 x amount of Chlorophyll b) 2.4 PROTEIN ASSIGNMENT The amount of protein in the leaves was determined by Bradford method (Bradford, 1976). In order to deter- mine the amount of protein, 0.25 g leaf tissue was ho- mogenized in 2.5 ml of 50 mM KH2PO4 (pH = 7) buffer in porcelain mortar. homogenate was transferred to ep- pendorf tubes and centrifuged at 15000 x G for 20 min- utes at +4 °C. 20 μl of supernatant was added in 2.5 ml of Coomassie brilliant blue G-250 and vortexed. After 10 minutes of incubation, the absorbance at 595 nm was measured and the amount of protein in the leaves was determined by means of a standard graphic. 2.5 PROLINE ASSIGNMENT To determine the amount of proline, 0.4 grams of leaf was homogenized in 4 % sulfosalicylic acid and then filtered through homogenate filter paper. 0.5 ml was tak- en from the filtrate and diluted 10 times with distilled water. 1 ml of sample, 1 ml of 96 % glacial acetic acid and 1 ml of acid ninhydrin solution were added to the tubes, and all the tubes were allowed to incubate for 60 min- utes in a water bath at 100 °C. After incubation, the tubes were held for 10 minutes in ice cubes, and then 2 ml of toluene was added to each tube and vortexed. After this process, five minutes were waited and the absorbance of the pink phase formed at the upper part of each tube was measured at 520 nm and recorded. The results were cal- culated as the amount of prolin per gram of fresh tissue using standard graphics prepared from pure proline. 2.6 MALONDIALDEHYDE ASSIGNMENT 1 g of plant tissue was homogenized with 0.1 % (w / v) TCA and centrifuged at 10000 x G for 5 minutes. Acta agriculturae Slovenica, 120/3 – 2024 5 Biochemical and molecular characterization of bread wheat genotypes under drought stress: Implications for antioxidant defense TCA containing 0.5 % TBA was added to the supernatant and incubated at 95 °C for 30 minutes and then quickly cooled in ice bath. The mixture was centrifuged again at 10000 x G for 15 minutes. The amount of malondialde- hyde was determined by measuring the absorbance of the supernatant at 532 nm in the spectrophotometer. 2.7 DNA EXTRACTION AND PCR AMPLIFICA- TION Young leaves reaching the two-leaf stage were used for DNA extraction, and it was performed according to the method of Doyle and Doyle (1990) with some modi- fications. The SSR primers used for this study were select- ed from the SSR primers that showed polymorphic prop- erties according to the previous drought characterization study (Ateş Sönmezoğlu & Terzi, 2018). PCR conditions used for Xwmc 89, Xwmc 118, Xwmc 304 and Xgwm 337 primers are as specified in the source article. Agarose gel concentrations were 2 % for PCR prod- ucts and 1 % for genomic DNAs. Ethidium bromide (10 mg ml-1) was used as an imaging dye in electrophoresis. Electrophoresis was performed in 1 % TBE buffer at 100 V constant for 60 min. PCR reactions were carried out with BIO-RAD C1000 Touch Thermal Cycler. Biorad ChemiDoc MP was used as a gel imaging system, and polymorphic bands were determined. 2.8 STATISTICAL ANALYSIS Polymorphic bands obtained from PCR were scored 1 (presence) and 0 (no band) for molecular characteriza- tion. DendroUPGMA (D-UPGMA) software (//genom- es.urv.es/UPGMA) was used for the molecular compari- sons of genotypes, and Interactive Tree of Life (iTOL) (https://itol.embl.de) was used to construct the interac- tive tree. 3 RESULTS AND DISCUSSIONS 3.1 ANTIOXIDANT ENZYME ACTIVITIES In Bayraktar, Sönmez, Gerek and İzgi cultivars, the amount of H2O2 and CAT varied inversely according to the stress days, while in Sultan, Harmankaya, Karahan, Kate and Sarıçanak cultivars, both were parallel. In this case, H2O2 produced as a result of stress factors was tried to be kept under control by CAT, but these plants had a Figure 2: Antioxidant enzyme activities (Since there is not enough vegetative tissue left in the Sarıcanak vari- ety, the analyzes on the 10th day could not be studied) Figure 3: Heatmap analysis of the antioxidant enzyme activities (Since there is not enough vegetative tissue left in the Sarıcanak variety, the analyzes on the 10th day could not be studied). Acta agriculturae Slovenica, 120/3 – 20246 N. ECZACIOGLU et al. phyll a, b and total chlorophyll amounts in parallel with the increasing stress conditions. (Fig.4, Fig. 5). 3.3 PROTEIN Drought tolerance of plants is usually achieved by changes in antioxidant enzymes and maintenance of H2O2 levels within certain limits. Water stress is associat- ed with increased oxidative stress due to increased accu- mulation of ROS, especially O2- and H2O2 in chloroplasts, mitochondria and peroxisomes. Induction of antioxidant enzyme activities is a general adaptation strategy used by plants to overcome oxidative stresses (Fig. 6) (Foyer & Noctor, 2003). In plants subjected to drought stress, changes in the amount of hydrophilic total protein within the cells were observed as a response to drought. A statistically signifi- cant increase in the amount of protein was observed in 7 out of 10 different varieties (Altay, Bayraktar, Gerek, Sultan, İzgi, Karahan and Kate) analyzed. This increase is thought to have been provided by the activities in the little more difficulty in maintaining the balance than the others. ASPX activity tended to decrease in Altay variety due to stress, while it increased in other wheat varieties compared to the control group. On the other hand, poly- phenol oxidase activity increased until the middle of the stress application in the other varieties except Sarıçanak variety, while it showed a decreasing trend as of the 10th day due to the deterioration of the internal balance (Fig. 2).While an increase in POD activity was observed in Bayraktar, Sultan, Izgi and Sarıçanak varieties, no signifi- cant change in POD activity was observed in other treat- ment groups due to stress (Fig. 3). 3.2 PHOTOSYNTHETIC PIGMENTS Photosynthesis is one of physiological processes, which is the most sensitively affected by abiotic stress conditions in plants. Drought stress also causes struc- tural and functional effects on chloroplasts. In this study, changes in chlorophyll a, b, total chlorophyll and carot- enoid contents were investigated in different wheat va- rieties under water stress. While no significant pigment change was observed in the samples during the early drought period, a relative decrease occurred in chloro- Figure 4: Photosynthetic pigment analysis (Since there is not enough vegetative tissue left in the Al- tay, Bayraktar, Gerek, Izgı, Sarıcanak, Sultan varie- ties the analyzes on the 10th day could not be studied) Figure 5: Heatmap analysis of the photosynthetic pig- ments (Since there is not enough vegetative tissue left in the Altay, Bayraktar, Gerek, Izgı, Sarıcanak, Sultan varie- ties the analyzes on the 10th day could not be studied) Acta agriculturae Slovenica, 120/3 – 2024 7 Biochemical and molecular characterization of bread wheat genotypes under drought stress: Implications for antioxidant defense synthesis of antioxidant enzymes, which are one of the intracellular defense systems. On the other hand, while no significant change was observed in Harmankaya va- riety, in Sönmez and Sarıçanak varieties, the plant was out of the hemostasis state and in parallel with this, a de- crease in total protein amount was observed. 3.4 PROLINE In the present study, proline levels increased signifi- cantly in all treatment groups except Altay and Sarıçanak, especially on the 10th drought day. This indicates that the plants were stressed after drought and this provided a sufficient signal for the activation of many biochemical and physiological antioxidant defense mechanisms with- in the organism. In Altay, the amount of free proline in- creased about five times after the 7th treatment day com- pared to the control group; however, it decreased back to its previous level by the 10th day. A similar situation was observed in Sarıçanak varietal as of the 3rd day (Fig. 7). 3.5 MALONDIALDEHYDE In Altay varietal, the amount of MDA, which reached its highest level on the 7th day, decreased on the 10th day. In this case, it is thought to be an indication that especially membrane lipids started to abound due to stress and the hemostasis state of the plant gradually started to disappear. 3.6 ANALYSES OF MOLECULAR CHARACTERS In this study, the dendrogram of the genotypes was conducted by using 4 SSR primers (Xwmc 89, Xwmc 118, Xwmc 304, and Xgwm 337), which show poly- morphic features in drought-related gene regions (Ateş Sönmezoğlu & Terzi, 2018; Kirigwi et al., 2007; Kumar et al., 2012; Mdluli et al., 2020). According to Figure 8, bread-wheat genotypes were divided into two main groups. While the first of these main groups were the Sultan-95 genotype, which is sensitive to drought, the second main group included the Gerek-79 genotype, which is resistant to drought. Group 1 contained the Sul- tan-95 and Karahan genotypes, while the other group contained all remaining genotypes. The second group was also divided into subgroups within itself. According to the dendrogram values, the closest genotypes to each other were Kate-1 and Sönmez 2001. The results of this study showed that the genotypes used have a high genetic diversity. Figure 6:Total protein, proline, MDA and H2O2 levels of treat- ment groups (Since there is not enough vegetative tissue left in the Altay, Bayraktar, Gerek, Izgı, Sarıcanak, Sultan varieties the H2O2 analyzes on the 10th day could not be studied) Figure 7: Heatmap analysis of al protein, Proline, MDA and H2O2 levels (Since there is not enough vegetative tissue left in the Altay, Bayraktar, Gerek, Izgı, Sarıcanak, Sultan varieties the H2O2 analyzes on the 10th day could not be studied) Acta agriculturae Slovenica, 120/3 – 20248 N. ECZACIOGLU et al. The antioxidant response is often enhanced in re- lation to plant tolerance when abiotic stress conditions occur because enhanced antioxidant enzyme activities contribute to drought tolerance to reduce oxidative dam- age (Gill & Tuteja, 2010). It has been stated in the litera- ture that high antioxidant enzyme activities contribute to stress tolerance (Mehrabad Pour-Benab et al., 2019), but different studies may show different results (Cruz de Carvalho, 2008). In this study, catalase enzyme activity increased gradually from day 0 in the tolerant genotype (Ger- ek-79), while it decreased significantly after the 3rd day for the sensitive genotype (Sultan-95). Catalase activities increased on the 10th day for Altay 2000, Harmankaya 99, and Izgi 2001 varieties. While the peroxidase enzyme activity did not show a big difference for Gerek-70, it de- creased and then increased for the Sultan on the 3rd day. Izgi 2001 and Altay 2000 cultivars were also reduced on the 3rd day the other days, like the Sultan-95. Ascorbate peroxidase activity decreased on the 3rd day for Gerek-79 but increased for the following days, it decreased on the 10th day in the Sultan-95 cultivar. Bayraktar, Altay 2000, Sönmez 2001 and Karahan also decreased on the 10th day. When evaluated biochemically, some of the wheat genotypes selected in different genotypes under drought stress are more resistant, and some are more sensitive. However, this varies for different biochemical analyzes. Therefore, no significant correlation was observed be- tween genotypes when the biochemical results were ex- amined in our study. Vuković et al. (2022) did not find a linear correla- tion between the results on the biochemical and molec- ular responses of winter wheat varieties under drought stress. They stated that the mechanism of drought toler- ance is quite complex. Accordingly, factors such as the period of the plant and the intensity or duration of the stress to which the plant is exposed should be considered (Vuković et al., 2022). Since genotypes under drought stress exhibit vari- ous biochemical properties, it is not enough to generalize the molecular data according to biochemical results. The reason for this can be explained as follows: 1) wheat has a large genome (16,000 Mb for bread wheat), and drought is a quantitative trait controlled by many genes. In this regard, the molecular markers may not be specific to the gene regions controlling the relevant biochemical pro- cesses; 2) molecular markers can also cover non-coding regions of the genome. 4 CONCLUSIONS In this study, the characterization of different wheat genotypes for drought tolerance as well as detailed bio- chemical analyzes, was evaluated. The tolerance mecha- nism of drought, which has a complex mechanism, was examined in terms of different genotypes. This study is expected to provide molecular and biochemical prelimi- nary information for local wheat varieties in our country. In addition, we propose a detailed molecular, biochemi- cal, and morphological examination of the drought mechanism for essential crop plants with subsequent studies. 5 ACKNOWLEDGEMENT This work was supported by the Karamanoglu Me- hmetbey University Unit for Scientific Research Projects, project no: 31-M-16. Figure 8: Dendrogram based on molecular data. Acta agriculturae Slovenica, 120/3 – 2024 9 Biochemical and molecular characterization of bread wheat genotypes under drought stress: Implications for antioxidant defense 6 REFERENCES Ahmad, P., Sarwat, M., & Sharma, S. (2008). Reactive oxygen species, antioxidants and signaling in plants. Journal of Plant Biology, 51, 167-173. Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology, 24(1), 1. Arora, N. K. (2019). 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