Acta agriculturae Slovenica, 120/4, 1–11, Ljubljana 2024 doi:10.14720/aas.2024.120.4.18648 Original research article / izvirni znanstveni članek Drought-induced expression of PvDERB1F and PvDREB5A with pro- moted antioxidant activities possibly enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) Motlalepula PHOLO-TAIT 1 , 2, Lekgari LEKGARI 3, and Moagisi ITHUTENG 3 Received April 26, 2024; accepted November 13, 2024 Delo je prispelo 26. april 2024, sprejeto 13. november 2024 1 Botswana University of Agriculture and Natural Resources-Faculty of Research and Graduate Studies, Research Centre for Bioeconomy, Gaborone-Botswana 2 corresponding author: mtait@buan.ac.bw 3 National Agricultural Research and Development Institute, Gaborone-Botswana Drought-induced expression of PvDERB1F and PvDREB5A with promoted antioxidant activities possibly enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) Abstract: The common bean (Phaseolus vulgaris L.) is an important source of protein, fiber, vitamins, and minerals, making it essential for food programs in Botswana. Prioritizing its integration into diversified farming is crucial for achieving social, environmental, and economic benefits. Previous stud- ies primarily focused on performance under rainfed, while the effect of drought stress remains unclear. The study aimed at evaluating the effect of drought stress on four (4) genotypes: DAB541, DAB515, CAL96, and GK011, while Tepary serves as the control. The study identifies CAL96 and DAB541 genotypes as the most promising genotypes for drought tolerance as dem- onstrated by their increased biomass production. The increase in biomass production may be due to the overexpression of the Phaseolus vulgaris dehydration-responsive element binding (PvDREB) genes, namely PvDREB1F and PvDREB5A. Higher proline and lower malondialdehyde (MDA) levels correlated with increased catalase (CAT) and ascorbate peroxidase (APX), which are linked to hydrogen peroxide (H₂O₂) scavenging ac- tivity. Conversely, GK011 and DAB514 exhibited decreased dry biomass, downregulated PvDREB1F, PvDREB5A, and Pv- DREB6B, along with greater levels of MDA and H₂O₂ and a steady activity of APX and CAT. This suggested an enhanced membrane lipid peroxidation and a loss of membrane integrity. Key words: Phaseolus vulgaris, drought stress, DREB genes, lipid peroxidation, antioxidants, ROS scavenging S sušo vzpodbujenim izražanjem genov PvDERB1F in Pv- DREB5A morda lahko s povečano antioksidacijsko aktivno- stjo povečamo toleranco navadnega fižola (Phaseolus vulga- ris L.) na sušni stres Izvleček: Navadni fižol (Phaseolus vulgaris L.) je pomem- ben vir beljakovin, vlaknin, vitaminov in mineralov, zaradi česar je bistven v programih prehane v Botswani. Njegovo prednostno vključevanje pri povečavanju raznolikosti kmeti- jske pridelave je bistveno za doseganje socialnih, okoljskih in ekonomskih ciljev. Predhodne raziskave so se prvenstveno usmerjale na njegovo uspevanje v razmerah namakanja z dežjem med tem, ko je ostajal učinek sušnega stresa nepojas- njen. V tej raziskavi so bili ovrenoteni učinki sušnega stresa na štiri genotipe in sicer DAB541, DAB515, CAL96 in GK011, pri čemer je ‘Tepary’ služil kot kontrola. V raziskavi sta bila genotipa CAL96 in DAB541 prepoznana kot najbolj obetajoča glede tolerance na sušo, kar se je pokazalo v njuni povečani izgradnji biomase. Povečana tvorba biomase bi lahko bila zaradi močno povečanega izražanja genov v fižolu, odzivnih na dehidracijo (PvDREB), kot sta gena PvDREB1F in PvDRE- B5A. Večja vsebnost prolina in manjša vsebnost malondial- dehida (MDA) sta soupadali v povečanjem aktivnosti kata- laze (CAT) in askorbat peroksidaze (APX), kar je povezano z odstranjevanje vodikovega peroksida (H₂O₂). Nasprotno sta genotipa GK011 in DAB514 pokazala zmanjšanje suhe biomase, zmanjšano izražanje PvDREB1F, PvDREB5A, in PvDREB6B genov s hkratnim povečanjem vsebnosti MDA in H₂O₂ in enakomerno aktivnostjo APX in CAT. To nakazuje povečano peroksidacijo membranskih lipidov in izgubo de- lovanja membran. Ključne besede: Phaseolus vulgaris, sušni stres, DREB geni, peroksidacija lipidov, antioksidati, ROS odstranjevanje Acta agriculturae Slovenica, 120/4 – 20242 M. PHOLO-TAIT et al. 1 INTRODUCTION The common bean (Phaseolus vulgaris L.) is an im- portant grain legume that represents a valuable source of protein in the diet (Broughton et al., 2003). P. vulgaris has been hailed as one of the priority crops for integration into diversified agricultural systems in Botswana (Minis- try of Agriculture, 2023). This policy has been motivated by common bean being considered the most important food legume crop in the human diet, mainly for its rich protein, carbohydrates, vitamins, dietary fibre and min- erals (Beebe et al., 2013; Mangole et al., 2022). It was fur- ther stated that the common bean is consumed in hospi- tals and school feeding schemes, and it is also offered as part of the government’s supplementary feeding scheme for underage children at clinics nationwide (Mangole et al., 2022). Although it is important, production has been reported to be low, resulting in a high import bill. In that respect, various common bean genotypes from neighbou countries have been introduced. Previous research on the introduced genotypes has been focused on planting date, nutritional value and yield stability under rainfed conditions (Moatshe- Mashiqa et al., 2021; Molosiwa et al., 2019). In addition, drought stress is a factor of economic importance in common bean production in Botswana. Southern Africa Drought Resilience Initiative (SADRI) (2021) indicated that the 2018/19 drought season resulted in two-thirds of crop failure. Botswana is a southern African country with an arid to semi-arid climate, resulting in desert- like conditions and very variable rainfall patterns across about a third of the country. This is caused by drastic and rapid changes in the global climate that can occur dur- ing the common bean’s life cycle, such as initial seedling establishment, vegetative growth, flowering and/or grain filling are exacerbated (Rao et al., 2013). This, there- fore, calls for screening of the introduced genotypes for drought stress tolerance. The response to drought stress is mediated by subtle changes in gene expression that lead to changes in the composition of the plant transcriptome, proteome and metabolome and ultimately the phenotype (Ansari et al., 2017, 2018; Deeba et al., 2012; Lin et al., 2016). The adaptive strategies used by drought-tolerant plants are of major importance for the selecting and adopting crops with improved performance under erratic water deficit conditions, such as Botswana’s. Pholo-Tait et al. (2022), reported the role of Phaseolus vulgaris ALLANTONAISE (PvALN) in drought stress in common bean genotypes. Meanwhile, transcriptional factors, especially the de- hydration-responsive element binding (DREB) factor family members comprise critical targets for selection of crops that confer tolerance to abiotic stress. DREBs regu- lates gene expression through a mechanism that involves recognizing the dehydration responsive element (DRE), which consists of the conserved motif A/GCCGAC (Sa- kuma et al., 2002). Amongst the six subgroups (A-1- A-6) of DREB genes, the expression of DREB2 (A-2), DREB5 (A-5; RAP2.1) and DREB6 genes (A-6; RAP2.4) showed to be highly induced under drought stress in Arabidop- sis (ChunJuan & JinYuan, 2010; Nakashima, Ito, & Ya- maguchi-Shinozaki, 2009). In another study, overex- pression of GmDREB1 increased the drought resistance of transgenic soybeans while increasing yield (Chen et al., 2022). Similarly, transgenic tobacco lines expressing the transcription factor gene of the DREB 5-A subgroup of Ricinus communis L. (RcDREB1) showed improved growth, drought tolerance and higher pollen viability (do Rego et al., 2021). Subsequent research showed that under drought stress conditions, the expression level of DREB1 rose more in wheat genotypes that were drought tolerant than in those that were drought sensitive (Rusta- mova et al., 2021). Previous studies reported that drought stress tolerance in the common bean is due to the over- expression of Phaseolus vulgaris DREB genes such as PvDREB1F, PvDREB5A and PvDREB6B (Konzen et al., 2019). Therefore, these DREB genes offer the possibility of serving as a basis for screening different genotypes of common beans for drought tolerance. Interestingly, the differential expression of DREB genes has been shown to also alter the functional expres- sion of nonenzymatic antioxidant defense (Ghaffari et al., 2019; Moloi & van der Merwe, 2021; Wei et al., 2016). The overexpression of DREB genes has been reported to induce a decrease in malondialdehyde (MDA) con- tent, and such an inverse correlation plays a vital role in drought stress tolerance. MDA is one of the final prod- ucts of polyunsaturated fatty acid peroxidation in cells. It is widely used as a reliable marker for determining the degree of membrane damage in tissues under stress and the ability of plants to tolerate drought stress (Blokhina et al., 2003; Morales & Munné-Bosch, 2019; Vendruscolo et al., 2007). The production of reactive oxygen species (ROS) under drought stress correlated positively with an increase in MDA content, resulting in increased perme- ability of the plasma membrane and extravasation of the content of cells. This inevitably impairs the production of biomolecules, such as lipids, proteins, and nucleic acids (Kong et al., 2016; Ripullone et al., 2021). On the other hand, lower concentrations of MDA have been reported to be associated with lower production of ROS. In soy- bean, tomato and wheat (Amoah & Seo, 2021; Raja et al., 2020; Saruhan Guler & Pehlivan, 2016), low levels of MDA suggested lower production of ROS and ulti- mately a reduction in membrane damage under drought stress. The overexpression of DREB genes suppressed the Acta agriculturae Slovenica, 120/4 – 2024 3 Drought-induced expression of PvDERB1F and PvDREB5A ... enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) production of MDA, resulting in an improved reactive oxidative species (ROS) scavenging capability. This was demonstrated in transgenic Arabidopsis, where overex- pression of AtDREB1A resulted in a lower MDA content (Morales & Munné-Bosch, 2019). Similarly, it was ob- served that the regulation of antioxidant mechanisms by Arabidopsis thaliana DREB1A (AtDREB1A) was associat- ed with a reduction in MDA levels in peanut (Arachis hy- pogaea L.) under water deficiency (Bhalani et al., 2019). In that respect, the production of MDA has been used as a robust reliable marker for determining the degree of injury to a drought-stressed plant (Alché, 2019; Kong et al., 2016; Morales & Munné-Bosch, 2019). In addition to MDA, osmolytes such as proline play an important role in plants under drought stress. Pro- line protects plants through cellular osmotic adjustment, ROS detoxification, protection of membrane integrity, and enzyme/protein stabilization (Ghaffari et al., 2019). Drought stress significantly enhanced the accumula- tion of proline in the leaves of canola at the flower ini- tiation stage and pod filling stage under drought stress. This response justified that proline accumulation under drought stress is an adaptive response that enhances survival and tissue water status (Morsi et al., 2023). This response is ascribed to enhanced osmotic modifications to acclimatize to recompense for plant survival and, ac- cordingly, assist in tolerating drought stress (Morsi et al., 2023). Conversely, a negative correlation between proline levels and plant growth has been reported in common beans. Higher levels of proline induced by stress inhib- ited growth in highly drought-sensitive cultivars and as such proline has been proposed as a suitable large-scale screening biochemical marker for common bean under water deficit and salt stress (Arteaga et al., 2020). Like- wise, overexpression of the DREB gene has been shown to enhance proline accumulation (Nguyen et al., 2019). Transgenic Arabidopsis plants overexpressing DREB1A showed a positive correlation of increased accumula- tion of proline. Transgenic rice overexpressing Oryza sativa DREB1A (OsDREB1A) also accumulated proline in stressed and controlled conditions (Dubouzet et al., 2003). Similar results were also demonstrated in soybean, in which overexpression of the Glycine max DREB6 and DREB2 (GmDREB6; GmDREB2) genes increased proline accumulation and tolerance to drought stress (Nguyen et al., 2019; Pham et al., 2020). Furthermore, peroxidase and catalases are among the versatile enzymatic hydro- gen peroxide (H₂O₂) scavenging systems (Caverzan et al., 2012; Foyer & Shigeoka, 2011; Shigeoka, 2002), hence contributing to the regulation of redox homeostasis and signaling pathways (Sohag et al., 2020; Tyagi et al., 2021). Given that specific DREB genes induce tolerance to drought stress in P. vulgaris (Konzen et al., 2019), it is worth noting that genetic variability exists among genotypes. The study aimed to evaluate the physiologi- cal and molecular responses of introduced common bean genotypes for drought tolerance. The objectives were to evaluate both enzymatic and nonenzymatic antioxidant responses, as well as conducting transcriptional screen- ing for drought stress tolerance using DREB genes. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS AND TREATMENTS The study was conducted at Sebele Research Station (24° 34’25’’S and 25° 58’0’’E) under the growth cabinet growth conditions. The growth cabinet was set at a long- day photoperiod of 16 h light and 8 h dark with the tem- perature set at 30 °C (day) and 25 °C (night). The light intensity was set at 300 µE m-² s-¹ light, while the humidity was maintained at 60 %. The study was conducted on four (4) common bean varieties, namely, DAB541, DAB514, CAL96, and GK011 The choice of common genotypes was based on their performance in the previous stud- ies, demonstrating their potential in terms of promising previous results on production and productivity stabil- ity under rainfed, as well as nitrogen fixation capability under water deficit conditions (Molosiwa et al., 2019; Pholo-Tait et al., 2022). Tepary bean, which is the com- monly grown bean landrace in Botswana was used at the control. Seeds were directly sown on a mixture (1:2) of sterilized sandy soil and loose jiffy growth media. The ex- periment was subjected to a factorial complete random- ized design with six (6). replications. The two treatments consisted of a maximum water holding capacity (control) and a drought stress treatment in which water was with- hold for fourteen days (Nakashima et al., 2009). 2.2 DRY LEAF BIOMASS DETERMINATION Six (6) replicates of leaf plant material were har- vested at the end of the drought stress period. Samples were oven-dried at 65 °C) for three (3) consecutive days. Thereafter, the samples were weighed for total leaf dry biomass production. 2.3 PROLINE AND MALONDIALDEHYDE (MDA) CONTENTS Leaf tissue material (0.1 g) was ground in liquid nitrogen and homogenized in 3 % (w/v) aqueous sulfo- Acta agriculturae Slovenica, 120/4 – 20244 M. PHOLO-TAIT et al. salicylic acid. The homogenate was centrifuged at 4000 × g for 10 minutes, while the resulting supernatant was added to a mixture of equal volumes of acid-ninhydrin and acetic acid. The mixture was incubated at 98  °C for 30 minutes and cooled to 25  °C, followed by the addition of toluene. The upper layer of the solution was aliquoted and used for proline determination. The pro- line content was quantified by spectrophotometer at an absorbance rate of 520 nm and calculated based on the standard curve using proline as a standard (Bates et al., 1973). Malondialdehyde (MDA) content was measured to determine the degree of membrane lipid peroxidation (Zhang and Huang, 2013). The total ground leaf samples (0.1 g) were homogenized in 0.1 % (w/v) trichloroacetic acid (TCA). The homogenate was centrifugated at 10,000 × g for 10 min. The supernatant was mixed with 20  % TCA consisting of 0.5 % thiobarbituric acid (TBA). The mixture was then heated at 95 °C for 15 min, followed by cooling on ice. Cooled samples were centrifuged at 4800 rpm for 10 min, after which the absorbance was mea- sured at 450, 532 and 600 nm. 2.4 ACTIVITIES OF ANTIOXIDANT ENZYMES Leaf tissues (250 mg) were homogenized in 0.1  % (w/v) TCA, centrifuged at 12 000 × g for 15 min before the addition of 10 mM potassium phosphate buffer (pH 7.0) and 1 M potassium iodide (KI) to the supernatant. The enzyme activity was read at an absorbance rate of 390 nm, while the H₂O₂ concentration was calculated using a standard curve (Velikova et al., 2000). Ascorbate per- oxidase (APX) and catalase (CAT) were extracted from ground leaf tissue (0.2 g) in liquid nitrogen. The mate- rials were homogenized in a mixture of 0.2 M sodium phosphate buffer (pH 7.8) and 0.1 mM EDTA. Consecu- tively, the homogenate was centrifuged at 15000 × g for 20 minutes at 4 °C followed by APX and CAT enzymatic activity assays. APX enzymatic activity was assessed in a reaction mixture consisting of an extract, 1 M potassium phosphate buffer (pH 7.8), 10 mM hydrogen peroxide, and 10 mM ascorbate. The reaction mixture without an extract was used as a blank. The reaction was initiated with the addition of H₂O₂ at 25  °C room temperature. The oxidation rate of ascorbate was determined by the decrease in absorbance at 290 nm for 3 min. CAT enzymatic activity (Aebi, 1974) was performed in a reac- tion mixture containing 0.01 M H₂O₂ and 0.05 M potas- sium phosphate buffer (pH 7.0). The CAT enzyme was added to initiate the reaction, while the decrease in ab- sorbance at 240 nm during the initial 3 min was used to measure H2O2 activity. 2.5 REAL-TIME QUANTITATIVE ANALYSIS OF DREB GENES CTAB protocol (Hu et al., 2002) was followed to isolate total RNA from three biological replicates of leaf tissue material (250 mg). RNA was purified us- ing a Qiagen RNase-free DNase Kit (Cat #79254) and eluted in RNase-free water according to the manufac- turer’s instructions. RNA concentrations were checked using a NanoDrop ND-1000 UV–Vis Spectrophotom- eter (Thermo Fisher Scientific), while RNA integrity was validated by visualizing the RNA on a 1 % agarose gel. RNase-DNase free RNA was reverse transcribed to complementary DNA (cDNA) using an oligo (dT¹⁸) primer and M-MLV (H-) reverse transcriptase (Pro- mega, Anatech, South Africa) following the manufac- turer’s instructions. cDNA template was checked by RT‒PCR using reference genes, namely, the SKP1/ASK- INTERACTING PROTEIN 16 (PvSKIP16), ACTIN 11 (PvACT11), and TUBULIN BETA-8 (Pvβ-TUB8) genes (Borges et al., 2012), which serve as internal control genes (Table 1). Real-time PCR was performed to test for the rela- tive expression of the drought stress-related marker genes PvDREB1F, PvDREB5A and PvDREB6B (Konzen et al., 2019) on four bean genotypes to evaluate their tolerance to drought stress (Table 1). RNA templates were replicated thrice (3) and diluted to a concentra- tion of 10 ng µl-¹. The PCR experiment was conducted using a Luna Universal One-step RT‒qPCR Kit (New England Biolabs, USA) consisting of 1X Luna Universal One-Step Reaction Mix (10 µl), 1 x Luna WarmStart® RT Enzyme Mix (1 µl), 0.4 µM of each primer (0.8 µl), 1 µg of diluted RNA and RNase-free water. Triple-repli- cate RT-qPCRs were performed in 96-well plates using a LineGene 9600 Bioer PCR machine (Hangzhou Bioer Technology) following SYBR Green/FAM detection. The RNA template was reverse transcribed at 55 °C for 60 s, followed by initial denaturation at 95°C for 60 s, 40 cycles of denaturation at 95°C for 10 s and extension at 60 °C for 35 s, and a melting step at 95 °C. PCR efficiency (E) was calculated using LinRegPCR (version 2014.5), while threshold (Ct) values were used to determine the relative expression level of a given gene using the 2-ΔΔCt method (Livak & Schmittgen, 2001; Schmittgen & Li- vak, 2008). The relative expression of genes common bean genotypes was then compared to that of Tepary bean as the control. 2.6 STATISTICAL DATA ANALYSIS The data were subjected to analysis of variance Acta agriculturae Slovenica, 120/4 – 2024 5 Drought-induced expression of PvDERB1F and PvDREB5A ... enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) (ANOVA) using the statistical software SPSS (version 22 of Windows; SPSS). One-way analyses of variance fol- lowed by Tukey’s HSD test comparison at p ≤ 0.05 were performed to determine the relevant differences between the control and drought-stressed variants of the respec- tive genotype. 3 RESULTS 3.1 DRY LEAF BIOMASS IN RESPONSE TO DROUGHT Similarly, compared with that of the control plants, the growth rate of the drought-stressed plants in terms of dry biomass was not affected in the CAL96 or DAB541 genotype. Interestingly, drought stress significantly reduced dry biomass production in the GK011 and DAB514 genotypes. This response translated to signifi- cant dry biomass production inhibition of 0.68 g (28 %) and 0.94 g (33 %) in GK011 and DAB514, respectively (Table 2). 3.2 LIPID PEROXIDATION LEVELS AND PRO- LINE PRODUCTION The lipid peroxidation in leaves as determined by the MDA content, varied significantly between stressed and control plants for the GK011 tepary bean and DAB514 common bean. The most significant drought-induced increase in the accumulation of MDA of 5.06 µmol g-1 fresh mass (117.3 %) was demonstrated in tepary bean, followed by 2.75 µmol g-1 fresh mass (42.04  %) in the DAB514 genotype. However, compared with those in the control plants, the MDA content in the stressed plants was not significantly greater for CAL96 and DAB541 (Table 2). A significant increase in the biosynthesis of proline of 1.09 (23.6 %) and 1.70 (39.6 %) mg-1 fresh mass was in- duced in the CAL96 and DAB541 drought-treated plants, respectively, in comparison to their corresponding con- trol plants. However, drought stress significantly inhib- ited the production of proline by 0.94 mg-1 fresh mass (22 %) in GK011 tepary beans (Table 2). 3.3 ANTIOXIDANT ENZYMATIC ACTIVI- TIES The enzymatic activities varied between the stressed plants and the control plants in terms of H₂O₂ concen- tration were observed for all the genotypes except for the DAB514 genotype. Drought stress significantly pro- moted this enzyme activity by 5.43 µmol g-1 fresh mass (68.6 %) in GK011 tepary bean and 2.82 µmol g-1 fresh mass (47.1  %) in DAB514 common bean. The reverse was observed in CAL96, which exhibited a significant 2.5 µmol g-1 fresh mass (27 %) reduction in H₂O₂ enzymatic Reference genes (Borges et al., 2012) NCBI ID Gene Forward primer (5’- 3’) Reverse Primer (5’- 3’) Amplicon size (bp) 62703083 ACTIN-11 (PvACT11) TGCATACGTTGGT- GATGAGG AGCCTTGGGGTTAA- GAGGAG 190 171656465 TUBULIN BETA-8 (PvΒ-TUB8) AATGTGAAGTC- CAGCGTGTG CTTCCCCAGTGTAC- CAATGC 163 187434529 SKP1/ASK-INTER- ACTING PROTEIN 16 (PvSKIP16) CACCAGGATG- CAAAAGTGG ATCCGCTTGTCCCTT- GAAC 163 Biotic stress genes (Konzen et al., 2019) Phytozome accession ID Gene Forward primer (5’- 3’) Reverse Primer (5’- 3’) Amplicon size (bp) Phvulv091025959m.g PvDREB1F TGCGTCGAGCAATTA- GAGAA TCCTGATGCGTCTG- GTATTG 153 Phvulv091010162m.g PvDREB5A TTGGGTACTTTTCC- CACTGC GCCTTCCATGTCAT- CATCCT 177 Phvulv091016691m.g PvDREB6B AATTCTGCATCTCC- CTCACG GCTGGGCTTGATTTA- GACGA 167 Table 1: Reference and target genes for the real-time PCR-based relative expression of genes. Acta agriculturae Slovenica, 120/4 – 20246 M. PHOLO-TAIT et al. activity in response to drought stress (Fig. 2A). While drought stress increased APX activity in DAB514 (3.37 µmol mg-1 FM protein min-1 19,6 %), drought stress did not affect APX activity in the GK011 and CAL96 geno- types (Fig. 2B). Drought stress also induced variations in catalase activity between the stressed plants and the control plants. Although plants exposed to drought stress presented significant 2.71 µmol mg-1 FM protein min-1 (14.5 %) and 3.30 µmol mg-1 FM protein min-1 (15.4 %) increases in catalase activity in CAL96 and DAB541, re- spectively, such increases in enzyme activity in GK011 and DAB514 were not significant (Fig. 2C). 3.3 RELATIVE EXPRESSION OF DEHYDRATION- RESPONSIVE ELEMENT BINDING (DREB) GENES The qPCR analysis was conducted on a highly intact RNA template that showed clear gel bands correspond- ing to 18S and 28S rRNA and the absence of a smear (Fig. 2A). The first qPCR experiment on the GK011 tepary bean and CAL96 common bean genotypes demonstrated an increase in the relative expression of the PvDREB1F, PvDREB5A, and PvDREB6B genes in GK011 tepary bean plants. Drought stress induced at least a 1-fold decrease in the relative expression of PvDREB1F and PvDREB6B as well as a 2-fold decrease in PvDREB5A in GK011 tepary bean plants. Similar results were observed for the CAL96 genotype, in which the relative expression of PvDREB5B and PvDREB6B were inhibited. However, there was a significant 1.2-fold increase in the expression of DREB1F in drought-stressed plants compared to that in the corresponding control plants. (Fig. 2B). A study between GK011 tepary bean and DAB514 revealed the distinct suppression of the differential expression of all three DREB genes in both genotypes. The highest average levels of 1.7-fold and 1.3-fold inhibition of PvDREB5A relative expression were revealed in the GK011 and DAB514 genotypes, respectively. Taken together, these findings indicated that drought stress induced significant and marked downregulation of the differential expres- sion of PvDREB5A compared with that of the other two DREB genes in both the GK011 and DAB514 genotypes (Fig. 2C). A similar trend of a downregulated expres- sion of PvDREB1F, PvDREB5A, and PvDREB6B in the which DREB gene were analyzed in GK011 and DAB541. Amongst the three genes, PvDREB5A was highly differen- tially expressed (2.9-fold). Drought stress downregulated the relative expression of PvDREB1F and PvDREB6B in the DAB541 genotype. In contrast, a marked increase in the expression of PvDREB5A (1.9-fold) was detected in DAB541 drought-stressed plants compared with control plants (Fig. 2D). 4 DISCUSSIONS Climate-adaptive strategies such as the use of drought-tolerant plants in Botswana are highly impor- tant for the selection and introduction of crops with improved performance under fluctuating water defi- cit conditions. This study adopted the considerable ef- fort that is devoted to the selection of crops using plant morphophysiological parameters coupled with molecu- lar and biochemical selection approaches. Intriguingly, drought stress upregulated the differential expression of PvDREB1F, which might have contributed to the main- tenance of dry biomass production in the CAL96 com- mon bean genotypes. This finding was in agreement with that of a previous study on rice, which demonstrated the overexpression of OsDREB1F under salt, drought, and low-temperature tolerance (Wang et al., 2008). The in- duced overexpression of PvDREB1F was accompanied by an increase in proline and antioxidant enzymes in the present study. Interestingly, an increase in proline level positively correlated with the maintained levels of MDA, hence suggesting the prevention of lipid peroxidation. In addition, an increase in CAT activity suggested an enhanced CAT enzymatic antioxidative defense mecha- nism that plays a role in the detoxification of H₂O₂ there- by maintaining equilibrium (Apel & Hirt, 2004; Ghaffari Genotype Dry biomass (g) MDA content (mg-1 fresh mass) Proline (mg-1 fresh mass) Control Drought stress Control Drought stress Control Drought stress GK011 2.40 a 1.73 b 5.67 a 9.29 b 4.19 a 3.25 bc CAL96 2.77 a 2.76 c 10.99 b 10.06 b 4.60 a 5.69 e DAB514 2.86 a 1.92 b 6.95 ad 11.23 bc 3.82 b 3.48 bc DAB541 3.67 d 3.60 d 9.67 bcd 5.35 a 4.29 b 5.98 e Table 2. Dry biomass production and accumulation of metabolites in response to drought stress. Values are represented as the mean ± SEM (n = 6) of independent biological replications. Values followed by the same letter do not differ from each other by Tukey’s test (p ≤ 0.05). Acta agriculturae Slovenica, 120/4 – 2024 7 Drought-induced expression of PvDERB1F and PvDREB5A ... enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) Figure 1: The effect of drought stress on the accumulation of H2O2 (A), APX activity (B) and CAT activity in common bean geno- types. Mean values represent ± SEM (n = 6) of independent biological replications. Different lower-case letters indicate significant (p ≤ 0.05) differences between mean values according to Tukey’s tests made separately for each genotype for the drought stresses against the control. Figure 2: Relative expression of DREB genes run on high intact RNA template (A) for CAL96 (B), DAB514 (C) and DAB541 (D) common bean genotype in response to drought. Real-time PCR was conducted in the respective common bean genotypes against the GK011 tepary bean. Relative expression rates of DREB genes were normalized against three internal reference genes (PvACT11; PvTUB-β8; PvSKIP1/16). Bars represent the mean ± standard error of the mean (SE) of pooled three technical samples from three independent samples. Values sharing a common letter are not significantly different at p < 0.05. Acta agriculturae Slovenica, 120/4 – 20248 M. PHOLO-TAIT et al. et al., 2019; Gomes et al., 2022). The drought tolerance mechanism for CAL96 likely occurs through the expres- sion of PvDREB1F, maintenance of ROS homeostasis and prevention of lipid peroxidation-related cell death. These are additional proposed drought stress tolerant mecha- nisms that also support the previous study that indicated that CAL96 could confer drought tolerance through the promotion of allantoin pathways (Pholo-Tait et al., 2022). The expression of a DREB 5-A subgroup of tran- scription factor genes from castor bean in tobacco has been reported to be associated with drought tolerance (do Rego et al., 2021). Similarly, the overexpression of PvDREB5A in our study suggested the presence of tran- scriptionally related drought tolerance mechanisms that resulted in the maintenance of growth in terms of leaf dry biomass. Contrary to inhibited growth as a result of high levels of protein (Arteaga et al., 2020), the growth rate was not affected by an increase in proline in this study. In concert with the previous report (Porch et al., 2009), the increased proline levels might have acted as a component of signal transduction pathways that regu- late the overexpression of PvDREB5A. In addition to its adaptive role in mediating osmotic adjustment and pro- tecting subcellular structure, an increase in proline in the DAB541 genotype could have played a role in maintain- ing higher activities of CAT. The latter could have sub- sequently maintained a steady activity of CAT-enzyme scavenging of H₂O₂ reactive oxygen species (Chen et al., 2022; Molinari et al., 2007; Noctor et al., 2018). Further- more, the overexpression of PvDREB5A correlated with a reduction in MDA in DAB541 and this is in line with the findings of a study on maize (Moussa & Abdel-Aziz, 2008). High levels of proline could have contributed to the reduction in MDA levels (Soares et al., 2019). This promoted an inverse correlation along with an increase in CAT activity, suggested a reduced lipid peroxidation and improved redox buffering as a result of effective scav- enging of H₂O₂ (Dong et al., 2018). On the contrary to CAL96 and DAB541 genotypes, drought stress-induced suppression of the differential expression of PvDREB1F, PvDREB5A, and PvDREB6B in GK011 and DAB514 genotypes. The downregulated differential expression of genes positively correlated with greater levels of proline, MDA, and H₂O₂ in the GK011 and DAB514 genotypes. Contrary to a previous study on common bean (Arteaga et al., 2020), decreased levels of proline resulted in an inhibited growth GK011 bean genotype. Rather, the inhibited growth rate might have been attributed to the promoted lipid peroxidation and eventual cell death due to the high accumulation of MDA and levels and the promoted H2O2 content (Ghaffari et al., 2019; Sivakumar et al., 2000; Soares et al., 2019). Pre- vious reports indicated that an increase in MDA content resulted in cell membrane rupture, hence increasing membrane leakage in P. vulgaris L. (Zlatev et al., 2006), Avena species (Pandey et al., 2010) and wheat (Tatar & Gevrek, 2008). In that respect, the downregulated ex- pression of DREB genes and increased levels of MDA and H₂O₂ suggested a promoted disequilibrium between H₂O₂-related ROS production and H₂O₂ scavenging ac- tivity (Yang et al., 2020) due to the stable cooperative ac- tivities of APX and CAT (Apel & Hirt, 2004; Gomes et al., 2022). Such disequilibrium could have resulted in an oxidative burst due to lipid peroxidation and protein de- naturation (P. Sharma et al., 2012; Yang et al., 2020). This could have resulted in greater levels of oxidative damage possibly through enhanced membrane lipid peroxidation and loss of membrane integrity to withstand the cellular- level effects of water loss and ultimately caused cellular damage and death, hence inhibiting plant growth (Kong et al., 2016; Ripullone et al., 2021; V. Sharma et al., 2019). An increase in MDA content, APX activity and greater levels of H₂O₂ was accompanied by a decrease in dry biomass in DAB514. As previously discussed above, increased levels of MDA suggested an increase in lipid peroxidation and subsequently promoted plant cell dam- age. Despite an increase in APX activity, the promoted production of H₂O₂ substantiated the need for APX for cooperative ROS enzymatic scavenging activity. This is in concert with previous studies which reported that in- creased APX activity in Salvinia molesta D. Mitch. and Vallisneria natans (Lour.) H. Hara resulted in H2O2 ac- cumulation, lipid peroxidation, and subsequently de- creased growth rates (Gomes et al., 2022). 5 CONCLUSIONS The current study demonstrated that CAL96 and DAB541 plants are possibly tolerant to drought stress. The CAL96 common bean genotype could confer drought tolerance through the overexpression of PvDREB1F and enhance the scavenging mechanism that involves the active role of proline in maintaining lipid peroxidation and the cooperative scavenging of H2O2 by CAT and APX. In the DAB541 common bean genotype, drought stress tolerance is associated with the overexpression of PvDREB5A and increased levels of proline, which co- operatively play a major role in the suppression of lipid peroxidation through reduced levels of MDA, hence stabilizing the membrane. In addition, such drought tolerance could have been attributed to H₂O₂ enzymatic scavenging activity, in which increased CAT activity en- hanced the maintenance of steady H₂O₂ levels. This find- ing therefore supported a previous study (Pholo-Tait et al., 2022) that the CAL96 and DAB541 genotypes serve Acta agriculturae Slovenica, 120/4 – 2024 9 Drought-induced expression of PvDERB1F and PvDREB5A ... enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) as promising drought-tolerant common bean genotypes. However, future reverse genetic approach studies that involve silencing PvDREB1F and PvDREB5A will un- ambiguously conclude their drought-induced tolerance role. This will further substantiate the importance of the inherent genotypic traits to serve as potential parent ma- terial in marker-assisted breeding to improve common bean varieties for drought tolerance and stress-induced ROS 6 CONFLICTS OF INTEREST There is no conflict of interest regarding the manu- script. 7 DATA AVAILABILITY Original data could be obtained upon reasonable requests from corresponding author. 8 ACKNOWLEDGEMENTS In memory of Professor Jens Kossmann from Stel- lenbosch University-Institute for Plant Biotechnology for providing guidance and advice on molecular and genet- ics applications. 9 REFERENCES Aebi, H. (1974). Catalase. Methods of enzymatic analysis. Jour- nal of Food Lipids., 2, 673–684. https://doi.org/https://doi. org/10.1016/B978-0-12-091302-2.50032-3 Alché, J. de D. (2019). A concise appraisal of lipid oxidation and lipoxidation in higher plants. Redox Biology, 23, 101136. https://doi.org/10.1016/J.REDOX.2019.101136 Amoah, J. N., & Seo, Y. W. (2021). Effect of progressive drought stress on physio-biochemical responses and gene ex- pression patterns in wheat. 3 Biotech, 11(10). https://doi. org/10.1007/s13205-021-02991-6 Ansari, W. A., Atri, N., Singh, B., Kumar, P., & Pandey, S. (2018). Morpho-physiological and biochemical responses of musk- melon genotypes to different degree of water deficit. Pho- tosynthetica, 56(4), 1019–1030. https://doi.org/10.1007/ s11099-018-0821-9 Ansari, W. A., Atri, N., Singh, B., & Pandey, S. (2017). Changes in antioxidant enzyme activities and gene expression in two muskmelon genotypes under progressive water stress. Bio- logia Plantarum, 61(2), 333–341. https://doi.org/10.1007/ s10535-016-0694-3 Apel, K., & Hirt, H. (2004). Reactive oxygen species: Me- tabolism, oxidative stress, and signal transduction. An- nual Review of Plant Biology, 55(1), 373–399. https://doi. org/10.1146/annurev.arplant.55.031903.141701 Arteaga, S., Yabor, L., Díez, M. J., Prohens, J., Boscaiu, M., & Vicente, O. (2020). The use of proline in screening for tolerance to drought and salinity in common bean (Pha- seolus vulgaris L.) genotypes. Agronomy, 10(6). https://doi. org/10.3390/agronomy10060817 Beebe, S. E., Rao, I. M., Blair, M. W., & Acosta-Gallegos, J. A. (2013). Phenotyping common beans for adaptation to drought. Frontiers in Physiology, 4 MAR. https://doi. org/10.3389/fphys.2013.00035 Bhalani, H., Thankappan, R., Mishra, G. P., Sarkar, T., Bosamia, T. C., & Dobaria, J. R. (2019). Regulation of antioxidant mechanisms by AtDREB1A improves soil-moisture deficit stress tolerance in transgenic peanut (Arachis hypogaea L.). PLoS ONE, 14(5). https://doi.org/10.1371/journal. pone.0216706 Blokhina, O., Virolainen, E., & Fagerstedt, K. V. (2003). Anti- oxidants, oxidative damage and oxygen deprivation stress: a Review. Annals of Botany, 91, 179–194. https://doi. org/10.1093/aob/mcf118 Broughton, W. J., Hernández, G., Blair, M., Beebe, S., Gepts, P., & Vanderleyden, J. (2003). Beans (Phaseolus spp.) - Model food legumes. Plant and Soil, 252(1), 55–128. https://doi. org/10.1023/A:1024146710611 Caverzan, A., Passaia, G., Rosa, S. B., Ribeiro, C. W., Lazzarotto, F., & Margis-Pinheiro, M. (2012). Plant responses to stress- es: Role of ascorbate peroxidase in the antioxidant protec- tion. Genetics and Molecular Biology, 35(4), 1011–1019. https://doi.org/10.1590/S1415-47572012000600016 Chen, K., Tang, W., Zhou, Y., Chen, J., Xu, Z., Ma, R., Dong, Y., Ma, Y., & Chen, M. (2022). AP2/ERF transcription fac- tor GmDREB1 confers drought tolerance in transgenic soybean by interacting with GmERFs. Plant Physiology and Biochemistry, 170, 287–295. https://doi.org/10.1016/j. plaphy.2021.12.014 ChunJuan, D., & JinYuan, L. (2010). The Arabidopsis EAR-mo- tif-containing protein RAP2.1 functions as an active tran- scriptional repressor to keep stress responses under tight control. BMC Plant Biology, 10(47), 1–15. Deeba, F., Pandey, A. K., Ranjan, S., Mishra, A., Singh, R., Shar- ma, Y. K., Shirke, P. A., & Pandey, V. (2012). Physiological and proteomic responses of cotton (Gossypium herbaceum L.) to drought stress. Plant Physiology and Biochemistry, 53, 6–18. https://doi.org/10.1016/j.plaphy.2012.01.002 do Rego, T. F. C., Santos, M. P., Cabral, G. B., de Moura Cip- riano, T., de Sousa, N. L., de Souza Neto, O. A., & Aragão, F. J. L. (2021). Expression of a DREB 5-A subgroup tran- scription factor gene from Ricinus communis (RcDREB1) enhanced growth, drought tolerance and pollen viability in tobacco. Plant Cell, Tissue and Organ Culture, 146(3), 493– 504. https://doi.org/10.1007/s11240-021-02082-7 Dong, C., Ma, Y., Zheng, D., Wisniewski, M., & Cheng, Z. M. (2018). Meta-analysis of the effect of overexpression of dehydration-responsive element binding family genes on temperature stress tolerance and related responses. Frontiers in Plant Science, 9, 713. https://doi.org/10.3389/ fpls.2018.00713 Dubouzet, J. G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E. Acta agriculturae Slovenica, 120/4 – 202410 M. PHOLO-TAIT et al. G., Miura, S., Seki, M., Shinozaki, K., & Yamaguchi-Shi- nozaki, K. (2003). OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant Journal, 33(4), 751–763. https://doi.org/10.1046/j.1365- 313X.2003.01661.x Foyer, C. H., & Shigeoka, S. (2011). Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiology, 155(1), 93–100. https://doi.org/10.1104/ pp.110.166181 Ghaffari, H., Tadayon, M. R., Nadeem, M., Cheema, M., & Razmjoo, J. (2019). Proline-mediated changes in antioxi- dant enzymatic activities and the physiology of sugar beet under drought stress. Acta Physiologiae Plantarum, 41(2). https://doi.org/10.1007/s11738-019-2815-z Gomes, M. P., Kitamura, R. S. A., Marques, R. Z., Barbato, M. L., & Zámocký, M. (2022). The role of H2O2-scavenging en- zymes (ascorbate, peroxidase and catalase) in the tolerance of lemna minor to antibiotics: Implications for phytore- mediation. Antioxidants, 11(1). https://doi.org/10.3390/ antiox11010151 Hu, C. G., Honda, C., Kita, M., Zhang, Z., Tsuda, T., & Mori- guchi, T. (2002). A simple protocol for RNA isolation from fruit trees containing high levels of polysaccharides and polyphenol compounds. Plant Molecular Biology Reporter, 20(1), 69–69. https://doi.org/10.1007/BF02801935 Kong, W., Liu, F., Zhang, C., Zhang, J., & Feng, H. (2016). Non- destructive determination of Malondialdehyde (MDA) distribution in oilseed rape leaves by laboratory scale NIR hyperspectral imaging. Nature Publishing Group, 6, 35393. https://doi.org/10.1038/srep35393 Konzen, E. R., Recchia, G. H., Cassieri, F., Gomes Caldas, D. G., Berny Mier Y Teran, J. C., Gepts, P., & Tsai, S. M. (2019). DREB genes from common bean (Phaseolus vulgaris L.) show broad to specific abiotic stress responses and dis- tinct levels of nucleotide diversity. International Journal of Genomics, https://doi.org/10.1155/2019/9520642. https:// doi.org/10.1155/2019/9520642 Lin, H. H., Lin, K. H., Syu, J. Y., Tang, S. Y., & Lo, H. F. (2016). Physiological and proteomic analysis in two wild tomato lines under waterlogging and high temperature stress. Jour- nal of Plant Biochemistry and Biotechnology, 25(1), 87–96. https://doi.org/10.1007/s13562-015-0314-x Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods, 25(4), 402–408. https://doi. org/10.1006/METH.2001.1262 Mangole, G., Ithuteng, M., Radikgomo, M., & Molosiwa, O. O. (2022). Challenges and opportunities in common bean pro- duction and marketing in Botswana: prospects and farmer’s perspectives. African Journal of Food, Agriculture, Nutri- tion and Development, 22(5). https://doi.org/10.18697/aj- fand.110.20660 Moatshe-Mashiqa, O. G., Mashiqa, P. K., & Molosiwa, O. O. (2021). Proximate and mineral nutrition of common bean genotypes as influenced by harvesting time. Journal of Agricultural Science, 14(1). https://doi.org/10.5539/jas. v14n1p85 Molinari, H. B. C., Marur, C. J., Daros, E., De Campos, M. K. F., De Carvalho, J. F. R. P., Filho, J. C. B., Pereira, L. F. P., & Vieira, L. G. E. (2007). Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): Osmotic adjustment, chlorophyll fluorescence and oxidative stress. Physiologia Plantarum, 130(2). https://doi. org/10.1111/j.1399-3054.2007.00909.x Moloi, M. J., & van der Merwe, R. (2021). Drought tolerance re- sponses in vegetable-type soybean involve a network of bio- chemical mechanisms at flowering and pod-filling stages. Plants, 10(1502), https:// doi.org/10.3390/plants10081502 Academic. https://doi.org/10.3390/plants10081502 Molosiwa, O. O., Pharudi, J., Seketeme, S., Mashiqa, P., & Chir- wa, R. (2019). Assessing yield stability and adaptability of Andean common bean genotypes in the semi-arid environ- ment of Botswana. African Journal of Agricultural Research, 14, 1593–1600. https://doi.org/10.5897/ajar2019.13988 Morales, M., & Munné-Bosch, S. (2019). Malondialdehyde: Facts and artifacts. Plant Physiology, 180, 1246–1250. htt- ps://doi.org/10.1104/pp.19.00405 Morsi, N. A. A., Hashem, O. S. M., El-Hady, M. A. A., Abd- Elkrem, Y. M., El-temsah, M. E., Galal, E. G., Gad, K. I., Boudiar, R., Silvar, C., El-Hendawy, S., Mansour, E., & Abdelkader, M. A. (2023). Assessing drought tolerance of newly developed tissue-cultured canola genotypes under varying irrigation regimes. Agronomy, 13(3). https://doi. org/10.3390/agronomy13030836 Moussa, H. R., & Abdel-Aziz, S. M. (2008). Comparative re- sponse of drought tolerant and drought sensitive maize genotypes to water stress. Australian Journal of Crop Sci- ence, 1(1), 519–528. Nakashima, K., Ito, Y., & Yamaguchi-Shinozaki, K. (2009). Up- date on abiotic stresses in arabidopsis and grasses transcrip- tional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiology, 149, 89–95. https://doi.org/10.1104/pp.108.129791 Nguyen, Q. H., Vu, L. T. K., Nguyen, L. T. N., Pham, N. T. T., Nguyen, Y. T. H., Le, S. Van, & Chu, M. H. (2019). Over- expression of the GmDREB6 gene enhances proline accu- mulation and salt tolerance in genetically modified soybean plants. Scientific Reports, 9(1). https://doi.org/10.1038/ s41598-019-55895-0 Noctor, G., Reichheld, J. P., & Foyer, C. H. (2018). ROS-related redox regulation and signaling in plants. In Seminars in Cell and Developmental Biology (Vol. 80, pp. 3–12). https://doi. org/10.1016/j.semcdb.2017.07.013 Pandey, H. C., Baig, M. J., Chandra, A., & Bhatt, R. K. (2010). Drought stress induced changes in lipid peroxidation and antioxidant system in genus Avena. Journal of Environmen- tal Biology, 31(4). Pham, T. T. N., Nguyen, H. Q., Nguyen, T. N. L., Dao, X. T., Sy, D. T., Le, V. S., & Chu, H. M. (2020). Overexpression of the GmDREB2 gene increases proline accumulation and toler- ance to drought stress in soybean plants. Australian Journal of Crop Science, 14(3), 495–503. https://doi.org/10.21475/ ajcs.20.14.03.p2173 Pholo-Tait, M., Kgetse, T., Tsheko, G. N., Thedi, O. T., Lethola, K., Motlamme, E. O., Ithuteng, M. I., & Ngwako, S. (2022). Genotypic variation in response to drought stress is asso- ciated with biochemical and transcriptional regulation of Acta agriculturae Slovenica, 120/4 – 2024 11 Drought-induced expression of PvDERB1F and PvDREB5A ... enhanced drought stress tolerance in Common bean (Phaseolus vulgaris L.) ureides metabolism in common bean (Phaseolus vulgaris L.). Acta Agriculturae Slovenica, 118(2), 1–9. https://doi. org/10.14720/aas.2022.118.2.2541 Porch, T. G., Ramirez, V. H., Santana, D., & Harmsen, E. W. (2009). Evaluation of common bean for drought tolerance in Juana Diaz, Puerto Rico. Journal of Agronomy and Crop Science, 195(5), 328–334. https://doi.org/10.1111/j.1439- 037X.2009.00375.x Raja, V., Qadir, S. U., Alyemeni, M. N., & Ahmad, P. (2020). Impact of drought and heat stress individually and in com- bination on physio-biochemical parameters, antioxidant responses, and gene expression in Solanum lycopersicum. 3 Biotech, 10(5). https://doi.org/10.1007/s13205-020-02206-4 Rao, I., Beebe, S., Polania, J., Ricaurte, J., Cajiao, C., Garcia, R., & Rivera, M. (2013). Can Tepary bean be a model for im- provement of drought resistance. African Crop Science Jour- nal, 21(4), 265–281. Ripullone, F., Via, B., Biancolillo, A., Luan qifuluan, Q., Yanjie Li, C., Zhang, Y., Luan, Q., Jiang, J., & Li, Y. (2021). pre- diction and utilization of malondialdehyde in exotic pine under drought stress using Near-Infrared Spectroscopy. Frontiers in Plant Science, 12, 1–9. https://doi.org/10.3389/ fpls.2021.735275 Rustamova, S., Shrestha, A., Naz, A. A., & Huseynova, I. (2021). Expression profiling of DREB1 and evaluation of vegeta- tion indices in contrasting wheat genotypes exposed to drought stress. Plant Gene, 25(July 2020), 100266. https:// doi.org/10.1016/j.plgene.2020.100266 Sakuma, Y., Liu, Q., Dubouzet, J. G., Abe, H., Yamaguchi-Shino- zaki, K., & Shinozaki, K. (2002). DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Com- munications, 290(3), 998–1009. https://doi.org/10.1006/ bbrc.2001.6299 Saruhan Guler, N., & Pehlivan, N. (2016). Exogenous low- dose hydrogen peroxide enhances drought tolerance of soybean (Glycine max L.) through inducing antioxi- dant system. Acta Biologica Hungarica, 67(2). https://doi. org/10.1556/018.67.2016.2.5 Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3(6), 1101–1108. https://doi.org/10.1038/nprot.2008.73 Sharma, P., Jha, A. B., Dubey, R. S., & Pessarakli, M. (2012). Reac- tive oxygen species, oxidative damage, and antioxidative de- fense mechanism in plants under stressful conditions. Jour- nal of Botany, 2012. https://doi.org/10.1155/2012/217037 Sharma, V., Goel, P., Kumar, S., & Singh, A. K. (2019). An apple transcription factor, MdDREB76, confers salt and drought tolerance in transgenic tobacco by activating the expression of stress-responsive genes. Plant Cell Reports, 38(2). https:// doi.org/10.1007/s00299-018-2364-8 Shigeoka, S. (2002). Regulation and function of ascorbate perox- idase isoenzymes. Journal of Experimental Botany, 53(372), 1305–1319. https://doi.org/10.1093/jexbot/53.372.1305 Sivakumar, P., Sharmila, P., & Pardha Saradhi, P. (2000). Pro- line alleviates salt-stress-induced enhancement in ribu- lose-1,5-bisphosphate oxygenase activity. Biochemical and Biophysical Research Communications, 279(2). https://doi. org/10.1006/bbrc.2000.4005 Soares, C., Carvalho, M. E. A., Azevedo, R. A., & Fidalgo, F. (2019). Plants facing oxidative challenges—A little help from the antioxidant networks. Environmental and Ex- perimental Botany, 161. https://doi.org/10.1016/j.envexp- bot.2018.12.009 Sohag, A. A. M., Tahjib-Ul-Arif, M., Polash, M. A. S., Belal Chowdhury, M., Afrin, S., Burritt, D. J., Murata, Y., Hos- sain, M. A., & Afzal Hossain, M. (2020). Exogenous Glu- tathione-Mediated Drought Stress Tolerance in Rice (Oryza sativa L.) is Associated with Lower Oxidative Damage and Favorable Ionic Homeostasis. Iranian Journal of Science and Technology, Transaction A: Science, 44(4), 955–971. https:// doi.org/10.1007/s40995-020-00917-0 Southern Africa Drought Resilience Initiative (SADRI). (2021). Drought Resilience Profiles | Botswana. In Cooperation in International Waters in Africa Program (CIWA). Tatar, Ö., & Gevrek, M. N. (2008). Influence of water stress on proline accumulation, lipid peroxidation and water content of wheat. Asian Journal of Plant Sciences, 7(4), 409–412. https://doi.org/10.3923/ajps.2008.409.412 Tyagi, S., Shumayla, Madhu, Singh, K., & Upadhyay, S. K. (2021). Molecular characterization revealed the role of catalases under abiotic and arsenic stress in bread wheat (Triticum aestivum L.). Journal of Hazardous Materials, 403. https://doi.org/10.1016/j.jhazmat.2020.123585 Vendruscolo, E. C. G., Schuster, I., Pileggi, M., Scapim, C. A., Molinari, H. B. C., Marur, C. J., & Vieira, L. G. E. (2007). Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat. Journal of Plant Physi- ology, 164(10), 1367–1376. https://doi.org/10.1016/J. JPLPH.2007.05.001 Wang, Q., Guan, Y., Wu, Y., Chen, H., Chen, F., & Chu, C. (2008). Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabi- dopsis and rice. Plant Molecular Biology, 67(6), 589–602. https://doi.org/10.1007/s11103-008-9340-6 Wei, T., Deng, K., Liu, D., Gao, Y., Liu, Y., Yang, M., Zhang, L., Zheng, X., Wang, C., Song, W., Chen, C., & Zhang, Y. (2016). Ectopic expression of DREB transcription factor, AtDREB1A, confers tolerance to drought in transgenic Salvia miltiorrhiza. Plant and Cell Physiology, 57(8), 1593– 1609. https://doi.org/10.1093/pcp/pcw084 Yang, J., Wang, H., Zhao, S., Liu, X., Zhang, X., Wu, W., & Li, C. (2020). Overexpression levels of LbDREB6 differen- tially affect growth, drought, and disease tolerance in pop- lar. Frontiers in Plant Science, 11. https://doi.org/10.3389/ fpls.2020.528550 Zlatev, Z. S., Lidon, F. C., Ramalho, J. C., & Yordanov, I. T. (2006). Comparison of resistance to drought of three bean cultivars. Biologia Plantarum, 50(3), 389–394. https://doi. org/10.1007/s10535-006-0054-9