Acta agriculturae Slovenica, 118/2, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2541 Original research article / izvirni znanstveni članek Genotypic variation in response to drought stress is associated with biochemical and transcriptional regulation of ureides metabolism in common bean (Phaseolus vulgaris L.) Motlalepula PHOLO-TAIT 1, 2, Thuto KGETSE 3, Gaone Nthabeleng TSHEKO 3, Olerato Tsotlhe THEDI 3, Katso LETHOLA 1, Ebenezer Oteng MOTLAMME 1, Moagisi Innocent ITHUTENG 1 and Samodimo NGWAKO 4 Received February 23, 2022; accepted May 29, 2022. Delo je prispelo 23. februarja 2022, sprejeto 29. maja 2022 1 Department of Agricultural Research, Ministry of Agricultural Development and Food Security, Gaborone, Botswana 2 Corresponding author, e-mail: pholom@webmail.co.za 3 Department of Chemistry & Forensic Sciences, Botswana International University of Science and Technology, Palapye, Botswana 4 Faculty of Research and Graduate Studies, Botswana University of Agriculture and Natural Resources, Gaborone, Botswana Genotypic variation in response to drought stress is asso- ciated with biochemical and transcriptional regulation of ureides metabolism in common bean (Phaseolus vulgaris L.) Abstract: Ureidic legumes such as common bean (Phaseoulus valgaris L.) plants export nitrogen from the nod- ules to shoots and leaves as ureides during symbiotic biological nitrogen fixation. Common bean gene encoding allantoinase (allantoin amidohydrolase, EC 3.5.2.5), is a key enzyme that catalyses the hydrolysis of allantoin to allantoic acid. It plays a role in ureide generation for export and ureide catabolism to generate a nitrogen source in sinks tissues. As such, one of the adaptive mechanisms of plants to drought stress, is associ- ated with ureides accumulation. To identify genetic variation of common bean in response to drought stress, changes in the expression of ALLANTONAISE (PvALN) gene and ure- ides content were examined in the leaf tissues of the three common bean genotypes (CAL96, DAB514 and DAB541) and one tepary bean genotype (Phaseolus acutifolius A.Gray). Amongst all the genotypes, the suggested drought susceptibil- ity in DAB514 common bean genotype, was probably attrib- uted to a repressed PvALN expression rate which were cor- roborated by an impaired ureides levels, and reduced plant growth. On contrary, drought stress induced an upregulated relative expression of PvALN coupled with an increase in al- lantoin and allantoate in DAB541 common bean genotype. In addition, the sustained plant growth in CAL96 was probably attributed to a steady amount of allantoin synthesized under drought stress. Taken together, DAB541 and CAL96 common bean genotypes are the promising genotypes with an induced upregulated transcriptional control of catabolism and/or bio- synthesis of ureides, hence potential genotypes for selection and introduction under Botswana semi-arid conditions. Key words: common bean; drought stress; ureides: al- lantonaise; allantoin; allantoate Genetska spremenljivost odziva navadnega fižola (Phaseo- lus vulgaris L.) na sušni stres je povezana z biokemičnim in transkripcijskim uravnavanjem presnove ureidov Izvleček: Ureidne stročnice kot je navadni fižol (Phaseo- ulus valgaris L.) transportirajo med simbiontsko vezavo dušik iz nodulov v liste kot ureide. Pri navadnem fižolu je pomem- ben gen, ki kodira alantoinazo (alantoin amidohidrolaza, EC 3.5.2.5), ključni encim, ki katalizira hidrolizo alantoina v alan- toinsko kislino. Ta ima pomembno vlogo pri tvorbi ureidov za njihov eksport in razgradnjo kot vir dušika v tkivih ponora. Pri rastlinah je eden izmed prilagoditvenih mehanizmov na sušni stres povezan s kopičenjem ureidov. Za določitev ge- netske variabilnosti navadnega fižola na sušni stres so bile analizirane spremembe v izražanju gena za alantoinazo, AL- LANTONAISE (PvALN) in vsebnosti ureidov v listnih tkivih pri treh genotipih navadnega (CAL96, DAB514 and DAB541) in enem genotipu ostrega fižola, Phaseolus acutifolius A.Gray. Med vsemi genotipi bi občutljivost genotipa DAB514 nava- dnega fižola verejtno lahko pripisali zavrtju izražanja gena PvALN, kar je bilo povezano z zmanjšano tvorbo ureidov in slabšo rastjo. V nasprotju je sušni stres vzpodbudil povečano izražanje tega gena, kar je bilo povezano s povečanjem vseb- nosti alantoina in alantoata pri genotipu DAB541. Dodatno bi ohranjeno rast genotipa CAL96 lahko pripisali stalni količini allantoina, ki se sintetizira med sušnim stresom. Zaključimo lahko, da sta genotipa navadnega fižola DAB541 in CAL96 obetajoča, z vzpodbujeno povečano transkripcijsko kontrolo katabolizma in/ali biosinteze ureidov, ki bi lahko služila kot potencial za izbor in uvajanje ustreznih genotipov v sušnih razmerah Botswane. Ključne besede: navadni fižol; sušni stres; ureidi; alan- tonaza; allantoin; alantoat Acta agriculturae Slovenica, 118/2 – 20222 M. PHOLO-TAIT et al. 1 INTRODUCTION Common bean (Phaseolus vulgaris L.) is an im- portant grain legume in the human diet due to its high nutritional properties, such as proteins, vitamins and minerals (Broughton et al., 2003). One of the major benefits of common beans in agriculture is their capac- ity to symbiotically fix atmospheric nitrogen through associations with soil nitrogen-fixing rhizobia, thus re- ducing the need to use nitrogen fertilizers (Coleto et al., 2014). As such, common bean plants are not dependent on nitrogen fertilization for growth due to their abil- ity to form symbioses with atmospheric di-nitrogen fixing bacteroid located in root nodules. Plant growth and productivity is dependent on the accessibility of the newly available fixed nitrogen from the root to the vegetative and reproductive plant tissues. Ureide allantoin and its degradation derivate al- lantoate are a group of soil heterocyclic nitrogen com- pounds that play an essential role in the assimilation, metabolism, transport, and storage of nitrogen in high- er plants (Smith & Atkins, 2002). They serve as the ve- hicle for storage and xylem transport of symbiotically fixed nitrogen from root to the shoot, and as such play a key role in nitrogen utilization in ureide-type legumes (Kohl et al., 1990; Smith & Atkins, 2002; Zrenner et al., 2006). Once delivered to sink tissues, allantoin is con- verted to allantoate, which in-turn can be broken down completely to glyoxylate, releasing four molecules of ammonia and two molecules of CO2. Genes encoding allantoinase (allantoin amidohydrolase, EC 3.5.2.5), ca- talysis the first step in the degradation of the ureide al- lantoin and the synthesis of allantoate, the second most prominent ureide. It is therefore unique in this pathway such that it plays a role in ureide generation for export from the nodules as well as ureide catabolism to gener- ate a nitrogen source in leaves and other nitrogen sinks (Muñoz et al., 2001; Watanabe et al., 2014; Werner et al., 2013). Adaptive mechanisms of plants to abiotic stresses such as drought, include changes in the expression of genes involved, biosynthesis of compatible osmolytes and scavenging systems for reactive oxygen species (Han et al., 2014; Hasegawa et al., 2000). The inhibi- tion of nitrogen utilization under drought stress, is proposed to be attributed to N-feedback regulation, in which ureides would be among the signaling molecules triggering the inhibition (Charlson et al., 2009; King & Purcell, 2005; Rachid Serraj, Vadez et al., 1999). The in- duction and activation of enzymes with a subsequent increased levels of intermediary metabolites, particu- larly ureides allantoin and allantoate play a vital role in plant responses and adaptation to abiotic stresses (Alamillo et al., 2010; Smith & Atkins, 2002) . In soy- bean, high ureides levels in shoots and leaves correlated with nitrogen fixation inhibition (Rachid Serraj, Vadez, et al., 1999). In Arabidopsis thaliana (L.) Heynh. mu- tant lacking ALLANTONAISE (ALN), high levels of al- lantoin metabolites were reported due to an activated allantoin biosynthetic genes and/or repression of ALN expression rate. The response suggested that ureide me- tabolism and accumulation contribute to the abiotic stress response, which is regulated, at least in part, at the transcriptional level. In addition, this implied a possible elevated drought stress tolerance, possibly by reducing oxidative damage. (Irani & Todd, 2016). The symbiotic nitrogen fixation showed to be ex- tremely sensitive to drought stress and this effect could result in decreasing N accumulation and yield of leg- ume crops (Serraj, 1999; Rachid Serraj, 2003). However, ureide-exporting legumes, such as common beans are more sensitive to drought stress due to rapid decline in nitrogen fixation compared to amidic ones (Purcell et al., 2004; R Serraj, 1999; Rachid Serraj, Vadez et al., 1999). On contrary, a variable degree of nitrogen fixa- tion inhibition due to drought stress was found among the bean genotypes. An increase in both mRNA levels and ALN activity with a concomitant increase in roots, shoots and leaves ureide levels in common bean in re- sponse to drought was attributed to an elevated syn- thesis of allantoate (Alamillo et al., 2010). Remarkably, other studies demonstrated a positive correlation be- tween suppressed nitrogen fixation and accumulation of ureides in stems and leaves of both sensitive and tol- erant genotypes. Further variability was associated with the rise in allantoate level coupled with an increase in ALLANTOINASE gene expression and enzyme activity in the most sensitive genotype, which increased after inhibition of nitrogen fixation, suggesting that ureides originate in vegetative tissues as a response to water stress, probably mediated by the induction of allantoin- ase (Coleto et al., 2014). The overreliance on erratic rain coupled with rela- tively poor soil quality has resulted in poor productiv- ity of crops in Botswana, making the agricultural sector most vulnerable to climate change (FANRPAN, 2017). Crop diversification such as the use of drought-tolerant legumes with enhanced nitrogen fixation ability and improved utilization of the newly fixed nitrogen to en- hance crop productivity crops has been hailed as one of the potential adaptive measures to mitigate climate change. As such, Botswana has considered the introduc- tion of common bean into the cropping system as one of the climate smart agriculture approaches, combating poverty, environmental degradation, and improving soil health. This was further justified by its high nutri- Acta agriculturae Slovenica, 118/2 – 2022 3 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) tive value and commercial benefits such as source of income for many rural household (Beebe et al., 2013; Molosiwa et al., 2019). However, information on the performance of the potential common bean genotypes for introduction, particularly nitrogen fixation and uti- lization capability and crop productivity under Botswa- na conditions remain elusive. Therefore, this study was conducted to identify the growth and genetic response of common bean genotypes under drought stress. Bio- chemical analysis of ureides-derived metabolites and transcriptional analysis of Phaseoulus vulguris ALLAN- TOINASE relative gene expression was conducted for the identification and selection of the best and promis- ing common bean genotypes in terms of nitrogen fixa- tion and utilization under drought stress. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS AND GROWTH CONDI- TIONS Common bean genotypes were selected based on their superior stability, adaptability and yield per- formance in the previous studies conducted at Sebele and Pandamatenga respectively (Molosiwa et al., 2019). These includes three common bean (Phaseolus vulgaris L.) genotypes (DAB541; DAB514; CAL96) and GK011 tepary bean (Phaseolus acutifolius A.Gray.; GK011), the latter being reported in previous studies as a drought tolerant bean. The experiment was conducted in a growth cabinet, in a randomized block design, with six replications, under a 16 h light/8 h dark photoperiod at 25 °C and a light intensity of 100-150 μmol photons m-2s-1. Plants were exposed to water holding treatment three (3) weeks after emergence. Drought stress ex- periment consisted of two treatments, namely drought stress treatment by withholding water application with a serious drought stress (35-45 % water holding capac- ity) and the control by ensuring maximum water hold- ing capacity by watering (70-80 %). 2.2 UREIDES ACCUMULATION: ALLANTOIN AND ALLANTOATE The determination of ureides allantoin and allan- toate were performed by differential analyses of glyoxy- late derivative according to published protocol (Lesca- no, 2020). Ureides were extracted from leaf 15 mg liquid nitrogen grounded leaf tissue samples by boiling it in 50 mM potassium phosphate buffer (pH 7.0). Homoge- nates were centrifuged at 18,000 xg for 25 min at 4 °C to ensure the absence of debris and a clear supernatant containing ureides. Six biological replicates of 100 μl aliquots of each sample were collected in three separate tubes for the measurement of endogenous glyoxylate, allantoic acid-derived glyoxylate and allantoin-derived glyoxylate. Glyoxylate is converted into glycolic acid- phenylhydrazone and then oxidized by ferricyanide in the presence of concentrated acid and phenylhydrazine to give red-colored 1,5-diphenylformazan. The absorb- ance of supernatants was measured using a spectropho- tometer at 535 nm. 2.3 TRANSCRIPTOMIC ANALYSIS OF DIFFER- ENTIALLY EXPRESSED GENES Total RNA was extracted using a Quick-RNA Miniprep Kit (Zymo Research Corporation, Irvine, CA, United States) as per the manufacturer’s protocol and treated with DNase1 (Zymo Research Corporation, Ir- vine, CA, United States). The quantity and quality of the isolated RNA were evaluated, respectively, using a Nan- oDrop ND-1000 UV–Vis Spectrophotometer (Thermo Fisher Scientific) and by 1 % electrophoresis agarose gels according to manufacturer’s instructions. The qual- ity of each cDNA and the RT-qPCR were checked per by using standard PCR reaction and the housekeeping gene PvACTIN-2 primers (Díaz-Leal et al., 2012) and PvALN (Table 1). These primer pairs were designed using GeneScript qPCR primer design (https://www. genscript.com/tools/pcr-primers-designer/advanced). Luna Universal qPCR Master Mix (New England Bio- lab Inc., MA, USA) and primers were used to determine RNA expression. The qPCR reactions were performed using triple replicates of cDNA samples in 96-well plates and performed on the LineGene 9600 (Hangzhou Bioer Techonology), following SYBR Green/FAM detection. Reactions were prepared in a total volume of 20 μl ac- cording to Luna® Universal qPCR Master Mix Protocol (M3003; New England Biolab Inc., MA, USA) contain- ing: 1x Luna Universal qPCR Master Mix, 10 μM of for- ward and reverse primer, 100 ng cDNA template and nuclease-free water. The PCR cycles consisted of 1 cycle of initial denaturing at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 40 s. The melting curve was obtained by applying increasing temperature from 60 to 90 °C. The relative fold change for Phaseou- lus vulgaris ALLANTOINASE (PvALN) was calculated using the 2−ΔΔCt method (Livak & Schmittgen, 2001), and normalized against the housekeeping PvACTIN-2 gene (Díaz-Leal et al., 2012). Acta agriculturae Slovenica, 118/2 – 20224 M. PHOLO-TAIT et al. 2.4 DATA ANALYSIS The data collected were subjected to analysis of variance (ANOVA) using MINITAB computer software program, significant means were separated using pair- wise Tukey comparison at p < 0.05. 3 RESULTS 3.1 PHYSIOLOGICAL BIOMASS RESPONSE To determine the response of common bean to drought stress, fresh biomass was determined from above ground plant tissues after watering was withheld for 10 consecutive days. Biomass for the three geno- types including GK011, CAL96 and DAB541 did not differ under normal water growth conditions. However, a significant reduced biomass was observed in DAB514 common bean genotypes compared to GK011 tepary bean as well as compared to the two common bean genotypes, namely, CAL96 and DAB541. Comparing drought stressed plants to their relative control plants demonstrated no significant variation in biomass in GK011 tepary bean and DAB541 common bean gen- otype. However, drought stress induced a 38.6 % and 38.7 % significant reduction in biomass in CAL96 and DAB514 respectively. Further biomass comparisons were made between drought stressed plants in all the genotypes. The result indicated a 42.6 % reduction in Gene Forward Primers Reverse Primer ACTIN-2 (PvActin-2) 5ˈ-TTGCTTTCAAGGAGGGGGTATGC-3ˈ 5ˈ-GGAGCTTGGAACCTTTCGGTGC-3ˈ ALLANOTONAISE (PvALN) 5ˈ-ACAAGCATGATGCAGGTGCTGTGA-3ˈ 5ˈ-TGCCTCCACGACATCGCACA-3ˈ Table 1: Primer pairs used to determine expression of genes Figure 1: Biomass of common bean genotypes (Phaseolus vulgaris L.) in response to drought stress. Biomass was measured unbve ground plat tissues of three common bean genotypes (CAL97; DAB514; DAB541) and GK011 tepary bean. The standard error of mean of three independent flow cells is indicated by the error bars (n = 6). Bars with different lowercase letters indicate significant differences (p < 0.05) Acta agriculturae Slovenica, 118/2 – 2022 5 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) biomass in drought stressed DAB514 plants relative to drought stressed GK011 tepary bean. In addition, a 35.6 % and a 50.6 % reduction in biomass in drought stressed DAB514 plants were observed in comparison to drought stressed CAL96 and DAB541 common bean plants respectively. Taken together, all comparisons demonstrated the existence of clear variation between DAB514 and DAB541 common bean genotypes in terms of biomass (Figure 1). 3.2 UREIDES ACCUMULATION IN RESPONSE TO DROUGHT STRESS To investigate the production of ureides-derived metabolites in response to drought stress, levels of al- lantoin and allantoate were measured in the leaves of drought stressed plants and control plants (Figure 2). The results were visualized using heat map (Figure 3), generated with MINITAB analytical software (version 21.1). The result demonstrated a significant increase in allantoin metabolite in CAL96 (50.1 %), DAB514 (45.5 %) and DAB541 (47.1 %) common bean genotypes compared to the GK011 tepary bean under normal wa- ter conditions. A significant 60.0 % and 23.8 % increase in allantoin accumulation between the plants under normal condition and drought stress was detected for GK011 tepary bean and DAB541common bean geno- types respectively. In contrast, allantoin content was not significantly affected between the control plants and the drought stressed DAB514 and CAL96 common bean genotypes. In respect to allantoate metabolite, the levels of allantoate were not significantly affected in CAL96 and DAB514 common bean genotypes compared to GK011 tepary bean under normal water growth condi- tions. The study further compared variation in allan- toate levels for the drought stressed plants compared to their relative control plants. The result exhibited 24.6 %, 26.5 % and 47.8 % of reduced allantoate levels for GK011 tepary bean and two common bean genotypes, namely, CAL96 and DAB514 in drought stressed plants compared to the control plants. In contrast, DAB541 common bean genotype elicited a 31.0 % significantly increased levels of allantoate in the drought stressed plants relative to their control plants. Taken together, the response of DAB541 common bean genotype under water stress showed a similar trend for both allantoin and allantoate. Thus, water stress induced a significant increase in both allantoin and allantoate metabolite lev- els (Figure 2). 3.3 RELATIVE GENE EXPRESSION OF UREIDE METABOLISM To assess the correlation between allantonaise ureide and changes in ALLANTONAISE (PvALN) rela- tive gene expression, both metabolic accumulation of allantonaise and relative PvALN gene expression was performed on the leaves of genotypes. To assess wheth- er the accumulation of ureides results from changes in the transcription of genes related to ureide metabolism, quantitative real time PCR was performed to determine the mRNA levels of genes coding for key enzymes in the synthesis of ureides, ALLANTONAISE (ALN). Expres- sion level of PvALN gene in the three replicates samples were normalized against the expression of ACTIN-2 as the internal control. According to the pairwise Tukey comparison, the relative expression of PvALN gene in water-deficit plants compared to the control plants was significantly depressed for all the common beans geno- types, except for DAB541. GK011 tepary bean showed the highest 7.7-folds reduction in the relative expression of PvALN. This decrease was however insignificantly different from DAB514 and CAL96 common bean gen- otypes, which also showed a decreased PvALN expres- sion rate by 3.2 and 5.4-folds respectively. Intriguingly, only DAB541 common bean genotype, showed an in- crease in the expression rate PvALN mRNA (1.2-folds) in the leaves of drought stressed plants relative to the control plants (Figure 4). 4 DISCUSSIONS Legumes are agronomically and economically im- portant in many cropping systems because of their abil- ity to assimilate atmospheric nitrogen and maintaining soil fertility. These are highly desirable traits to consider in the improvement of legume productivity for sustain- able agricultural practices (Serraj, 1999; Rachid Serraj, 2003, 2003; Rachid Serraj et al., 1999). Drought stress is one of the most important environmental factors that regulate plant growth and development and limit its production. Legumes exhibit reduction in nodulation and biological nitrogen fixation in response to drought stress (Pimratch et al., 2008). Accumulation of ureide compounds has been reported in several plant species under stress conditions, and a considerable number of research articles argue for a hindered rather than active ureide catabolism as the survival trait for plants sub- jected to periods of mild drought or salinity due to the Acta agriculturae Slovenica, 118/2 – 20226 M. PHOLO-TAIT et al. alternative prime stress signaling function of uric acid and allantoin. Figure 2: Ureides accumulation in response to drought stress. Ureides measurement consisted of allantoin (A) and allantoate (B) accumulation for pants under control (T0) and drought stress (T1). Ureides accumulation was measured on leaf tissues of three common bean genotypes (CAL97; DAB514; DAB541) and GK011 tepary bean. Bars with different letters are statistically different according to p < 0.05. The standard of mean of three independent flow cells is indicated by the error bars error (n = 6) Acta agriculturae Slovenica, 118/2 – 2022 7 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) The current study evaluated the response of three common bean genotypes to drought stress at both bio- chemical and transcriptional level. Firstly, the response of common bean genotypes under normal growth conditions were tested against tepary bean (Phaseolus acutifolius A. Gray), a relatively higher drought-tol- erant crop than common bean (Phaseoulus vulgaris) and serving as genetic resource for food and genetic enhancement of related legumes (Mwale et al., 2020). The insignificant growth rate in terms of biomass un- der normal growth condition was accompanied by a significant increase in ureide allantoin levels in CAL96, DAB514 and DAB541 common bean genotypes rela- tive to GK011 tepary bean. On contrary, the allantoate content was not affected in CAL96 and DAB514 when compared to GK011 tepary bean. Taken together, the normal growth rate of common bean genotypes com- pared to GK011 tepary bean might have been sustained by an enhanced assimilation and metabolism of nitro- gen, which is attributed increased levels of allantoin and a sustained level of allantoate. Taking into consid- eration the 16 hours day growth period in the current study, this results are consistent with Arabidopsis thali- ana studies, which indicated that allantoin ureide deg- radation is important for the growth and development during vegetative growth under long-day conditions (Takagi et al., 2018). The response of bean genotypes was further evalu- ated under drought stress by comparing plants under drought stress against their relative control ones. In- triguingly, all the common bean genotypes, including tepary bean revealed a similar trend of induced inhib- ited plant growth under drought stress. However, only DAB514 common bean genotype showed a significant reduced plant growth in drought stressed plants com- pared to their relative control plants. The impaired plant growth rate in DAB514 was positively associat- ed with the reduction in both allantoin and allantoate levels, with a concomitant induced down-regulated PvALN relative gene expression. This response pro- posed an impaired ureides degradation at transcrip- tional level, which inevitably negatively affected assimi- lation and use of fixed N and eventually plant growth in DAB514 common bean genotype under drought stress. This finding is contrary to reports that indicated that DAB514 common bean genotype as a stable and high Figure 4: Relative Phaseolus vulgaris ALLANTONAISE (PvALN) gene expression in common beans in response to drought stress. Relative gene expression was measured on leaf tissues of three common bean genotypes (CAL97; DAB514; DAB541) and GK011 tepary bean. Bars with different letters are statistically different according to p < 0.05. The standard of mean of three independent flow cells is indicated by the error bars error (n = 3) Acta agriculturae Slovenica, 118/2 – 20228 M. PHOLO-TAIT et al. yielding genotype under drought stress (Molosiwa et al., 2019). Our results implies that DAB514 common bean genotype is a drought-sensitive genotype pos- sibly due to an impaired ureides metabolism at both chemical and transcriptional level with a substantial reduced plant growth. Though similar results were ob- served in terms of a suppressed expression of PvALN coupled with low levels of allantoate, the plant growth rate was not affected in water stressed CAL96 common bean genotype. The suppressed expression of PvALN in CAL96 common bean genotype might be responsible for an impaired rate of degradation of allantoin and the synthesis of allantoate, subsequently owing to a steady amount of allantoin synthesized under drought stress. Water deficit also resulted in another notable in- crease in ureides allantoin and allantoate levels cou- pled with an induced upregulated relative expression of PvALN in DAB541 common bean genotype. This is in concert with studies on Arabidopsis, Phaseolus vul- garis, and Soybean which demonstrated an increase in shoot ureides under drought stress (Alamillo et al., 2010; Ladrera et al., 2007; Rachid Serraj, 2003; Vadez & Sinclair, 2001). This advocated for an increased tran- scriptional regulation of purine metabolism by PvALN, which in turn resulted in enhancing both the degra- dation of the ureide allantoin and the synthesis of al- lantoate (Alamillo et al., 2010; Coleto et al., 2014). This response suggested that ureide accumulation is a gen- eral response to drought stress and is regulated at the transcriptional level mainly through the induction of allantonaise degradation and the subsequent allantoate synthesis in DAB541 common bean genotype leaf tis- sues. 5 CONCLUSIONS The current study evaluated the response of com- mon bean genotypes to drought stress by assessing ureides metabolism at biochemical and transcriptional level coupled with the ultimate plant growth in terms of biomass. Overall results suggested a degree of ge- netical variation among common bean genotypes. The enhanced plant growth or maintained growth rate un- der drought stress in DAB541 and CAL96 common bean genotypes was probably due to an enhanced degradation of the ureide allantoin and the synthesis of allantoate metabolites. These findings suggested an enhanced ureide generation for export and ureide ca- tabolism to generate a nitrogen source in leaves under drought. Therefore, the study concludes that DAB541 and CAL96 common bean genotype are potential gen- otypes for selection and introduction under Botswana semi-arid condition. Molecular reverse genetic stud- ies can further be conducted to confirm ureides me- tabolism and crop performance of DAB541 and CAL96 common bean genotypes under drought stress. 6 ACKNOWLEDGEMENTS This research was conducted at the Biotechnology and Genetics laboratory, Department of Agricultural Research for providing, who provided both infrastruc- ture and financial capital for consumables and molec- ular reagents. Many thanks to Legumes and Oil Seed Unit for providing us with the seed materials. 7 REFERENCES Alamillo, J. M., Luís Díaz-Leal, J., Victoria Sánchez-Moran, M. A., & Pineda, M. (2010). Molecular analysis of ureide ac- cumulation under drought stress in Phaseolus vulgaris L. Plant, Cell and Environment, 33, 1828–1837. https://doi. org/10.1111/j.1365-3040.2010.02187.x 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 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 Charlson, D. V., Korth, K. L., & Purcell, L. C. (2009). Allantoate amidohydrolase transcript expression is independent of drought tolerance in soybean. Journal of Experimen- tal Botany, 60(3), 847–851. https://doi.org/10.1093/jxb/ ern332 Coleto, I., Pineda, M., Rodiñ, O. A. P., De Ron, A. ., & Alamillo, J. . (2014). Comparison of inhibition of N2 fixation and ureide accumulation under water deficit in four common bean genotypes of contrasting drought tolerance. Annals of Botany, 113, 1071–1082. https://doi.org/10.1093/aob/ mcu029 Díaz-Leal, J. L., Gálvez-Valdivieso, G., Fernández, J., Pineda, M., & Alamillo, J. M. (2012). Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L. Journal of Experimen- tal Botany, 63(11), 4095–4106. https://doi.org/10.1093/ jxb/ers090 FANRPAN. (2017). FANRPAN Policy Brief CLIMATE- SMART AGRICULTURE IN BOTSWANA. In Promoting a Conducive Policy Environment for a Food and Nutrition Secure Africa (Issue 2017). Irani, S., & Todd, C. D. (2016). Ureide metabolism under abiot- ic stress in Arabidopsis thaliana. Journal of Plant Physiolo- gy, 199, 87–95. https://doi.org/10.1016/j.jplph.2016.05.011 King, C. ., & Purcell, L. C. (2005). Inhibition of N2 fixation in soybean is associated with elevated ureides and ami- Acta agriculturae Slovenica, 118/2 – 2022 9 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) no acids. Plant Physiology, 137, 1389–1396. https://doi. org/10.1104/pp.104.056317 Kohl, D. H., Lin, J. J., Shearer, G., & Schubert, K. R. (1990). Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules. Plant Physiology, 94(3), 1258–1264. https://doi.org/10.1104/ pp.94.3.1258 Ladrera, R., Marino, D., Larrainzar, E., González, E. M., & Ar- rese-Igor, C. (2007). Reduced carbon availability to bacte- roids and elevated ureides in nodules, but not in shoots, are involved in the nitrogen fixation response to early drought in soybean 1[OA]. Plant Physiology, 145, 539–546. https://doi.org/10.1104/pp.107.102491 Lescano, I. (2020). Determination of ureides content in plant tissues. Bio-Protocol, 10(11), 1–9. https://doi.org/10.21769/ bioprotoc.3642 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. htt- ps://doi.org/10.1006/METH.2001.1262 Molosiwa, O. O., Pharudi, J., Seketeme, S., Mashiqa, P., & Chirwa, R. (2019). Assessing yield stability and adapt- ability of Andean common bean genotypes in the semi- arid environment of Botswana. African Journal of Agri- cultural Research, 14, 1593–1600. https://doi.org/10.5897/ ajar2019.13988 Muñoz, A., Piedras, P., Aguilar, M., & Pineda, M. (2001). Urea is a product of ureidoglycolate degradation in chickpea. Purification and characterization of the ureidoglycolate urea-lyase. Plant Physiology, 125(2), 828–834. https://doi. org/10.1104/pp.125.2.828 Mwale, S. E., Shimelis, H., Mafongoya, P., & Mashilo, J. (2020). Breeding tepary bean (Phaseolus acutifolius) for drought adaptation: A review. Plant Breeding, 139, 821–833. https:// doi.org/10.1111/pbr.12806 Pimratch, S., Jogloy, S., Vorasoot, N., Toomsan, B., Patanothai, A., & Holbrook, C. C. (2008). Relationship between bio- mass production and nitrogen fixation under drought- stress conditions in peanut genotypes with different lev- els of drought resistance. Journal of Agronomy and Crop Science, 194(1), 15–25. https://doi.org/10.1111/J.1439- 037X.2007.00286.X Purcell, L. C., Serraj, R., SinclairT.R., & DeA. (2004). Soybean N2 fixation estimates, ureide concentration, and yield re- sponses to drought. Crop Science, 44, 484–492. https://doi. org/10.2135/cropsci2004.4840 Serraj, R. (1999). Review article. Symbiotic N2 fixation response to drought. Journal of Experimental Botany, 50(331), 143– 155. https://doi.org/10.1093/jexbot/50.331.143 Serraj, Rachid. (2003). Effects of drought stress on legume symbiotic nitrogen fixation: Physiological mechanisms. Indian Journal of Experimental Biology, 41(10), 1136–1141. Serraj, Rachid, Sinclair, T. R., & Purcell, L. C. (1999). Symbi- otic N2 fixation response to drought. Journal of Experi- mental Botany, 50(331), 143–155. https://doi.org/10.1093/ jxb/50.331.143 Serraj, Rachid, Vadez, V., Denison, R. F., & Sinclair, T. R. (1999). Involvement of ureides in nitrogen fixation inhibition in soybean. Plant Physiology, 119(1), 289–296. https://doi. org/10.1104/pp.119.1.289 Smith, P. M. C., & Atkins, C. A. (2002). Purine biosynthesis. Big in cell division, even bigger in nitrogen assimilation. Plant Physiology, 128(3), 793–802. https://doi.org/10.1104/ pp.010912 Takagi, H., Watanabe, S., Tanaka, S., Matsuura, T., Mori, I. C., Hirayama, T., Shimada, H., & Sakamoto, A. (2018). Dis- ruption of ureide degradation affects plant growth and development during and after transition from vegetative to reproductive stages. BMC Plant Biology, 18(1), 1–16. https://doi.org/10.1186/s12870-018-1491-2 Vadez, V., & Sinclair, T. R. (2001). Leaf ureide degradation and N2 fixation tolerance to water deficit in soybean. Journal of Experimental Botany, 52(354), 153–159. https://doi. org/10.1093/jxb/52.354.153 Watanabe, S., Matsumoto, M., Hakomori, Y., Takagi, H., Shi- mada, H., & Sakamoto, A. (2013). The purine metabolite allantoin enhances abiotic stress tolerance through syner- gistic activation of abscisic acid metabolism. https://doi. org/10.1111/pce.12218 Watanabe, S., Matsumoto, M., Hakomori, Y., Takagi, H., Shi- mada, H., & Sakamoto, A. (2014). The purine metabo- lite allantoin enhances abiotic stress tolerance through synergistic activation of abscisic acid metabolism. Plant, Cell and Environment, 37(4), 1022–1036. https://doi. org/10.1111/pce.12218 Werner, A. K., Medina-Escobar, N., Zulawski, M., Sparkes, I. A., Cao, F. Q., & Witte, C. P. (2013). The ureide-degrading reactions of purine ring catabolism employ three amido- hydrolases and one aminohydrolase in arabidopsis, soy- bean, and rice. Plant Physiology, 163(2), 672–681. https:// doi.org/10.1104/pp.113.224261 Zrenner, R., Stitt, M., Sonnewald, U., & Boldt, R. (2006). Py- rimidine and purine biosynthesis and degradation in plants. Annual Review of Plant Biology, 57, 805–836. htt- ps://doi.org/10.1146/annurev.arplant.57.032905.105421