Comparison of Antioxidant Enzyme Activities between Tylosema esculentum and Tylosema fassoglense in Response to Water Deficit

1. Introduction

Drought is one of the most significant abiotic stress factors affecting crop production and water resources in South Africa [1]. Prolonged exposure of plants to drought leads to increased cellular levels of reactive oxygen species (ROS), which can be deleterious to cells if they accumulate beyond appropriate thresholds [2,3]. To abate oxidative stress, plants mobilize an array of antioxidant defense systems [4]. Tolerance to oxidative stress is mostly associated with increased antioxidant activity. Several studies have investigated the response of the plant antioxidant system to drought stress. However, such studies have mostly focused on common bean, soybean, cowpea, and mung bean for cultivated grain legumes [5,6,7,8,9,10]. The only study that investigated drought responses in the genus Tylosema was limited to the evaluation of physiological responses [11].

Leguminous crops are beneficial to human nutrition because they serve as a good source of protein, dietary fiber, vitamins, and minerals [12]. Marama bean (genus Tylosema) grows in the semi-arid tropical/sub-tropical regions of Southern and Eastern Africa [13]. Amongst the five different species of Tylosema that are endemic to sub-Saharan Africa [14], Tylosema esculentum (Burch.) A. Schreib. and Tylosema fassoglense (Schweinf). Torre & Hillc. are the predominant Tylosema species in South Africa [14,15,16,17,18,19]. These species grow in diverse soil types, some of which are sandy and have limited water-holding capacity, and they can withstand drought far better than most of the currently cultivated legumes [11]. Although T. esculentum grows in more arid environments compared to the tropical and subtropical climates favored by T. fassoglense, the latter has a wider range of distribution across diverse habitats and may thrive in regions or environments where T. esculentum struggles to adapt. T. fassoglense occurs in eastern and central tropical Africa, from Sudan and Ethiopia southwards to Angola and Namibia and the north-eastern part of South Africa, while T. esculentum is restricted to southern Africa [14]. T. esculentum mostly grows in sandy plains with altitudes ranging from 900 to 1100 m, while T. fassoglense can thrive in diverse ecosystems including open woodlands, forest edges, cultivated fields, clay, sandy or rocky soils, and occasionally in periodically flooded areas, occurring at altitudes ranging from 30 to 2100 m [14]. Marama bean pods produce one to four seeds per pod, with varying color and shape. These edible seeds are round and brownish-black or flattened and chestnut-brown in T. esculentum and T. fassoglense, respectively [17,20,21]. In addition to their nutritional importance, these plants also have medicinal properties [22]. Although marama beans have nutritional and medicinal importance, they are not recognized as a staple food because it is not cultivated but rather found in the wild and only consumed by a small percentage of the population in the regions of Africa where it grows. If domesticated and properly maintained, marama bean could be a key drought resilient crop that could assist in addressing the impacts of climate change on food security. A comparative study on nutrient composition indicated that T. fassoglense possesses higher mineral levels and protein content compared to T. esculentum [23]. Distinct medicinal uses of these two species were reported. For example, in southern Africa, local communities use T. esculentum as medicine to treat asthma and diarrhea [24,25]. T. fassoglense is used to improve postpartum recovery, and to treat hypertension related diseases and impotence [25,26]. Local communities in Kenya use the boiled tuber of T. fassoglense to treat infertility in women, epilepsy, renal disease, and cancer [27]. In Uganda, it was also reported that extracts from T. fassoglense tuber are used as a treatment for various types of cancer [28]. T. fassoglense could offer unique nutritional and medicinal benefits that may not be found in T. esculentum. Therefore, improving drought tolerance in T. fassoglense is crucial for unlocking its full potential to ensure sustainable utilization of its nutritional and medicinal properties, contributing to enhanced food security and human health. On these bases, our study aimed at investigating differences in the physiological and biochemical responses between T. esculentum and T. fassoglense to water deficit stress. Understanding these responses in the two species will provide valuable insights towards improving drought tolerance in T. fassoglense and potentially other leguminous crops.

2. Materials and Methods

2.1. Plant growth and treatment

Seeds of T. esculentum (Burch.) A. Schreib. and T. fassoglense (Schweinf). Torre & Hillc. were obtained from the wild in South Africa near Tosca for T. esculentum (-25°49'37.5"S 23°42'30.3"E) and near Tzaneen for T. fassoglense (-23°52'12.8"S 30°06'05.8"E). The seeds were stored at room temperature at the Plant Stress Tolerance Laboratory at the University of the Western Cape until further use. The seeds were surface sterilized using chlorine gas and germinated as described by [29]. Soil was prepared by moistening Promix Organic with tap water, followed by supplementation with nutrients as described by [29]. The initial water potential of the soil was adjusted to − 0.3 ± 0.025 MPa for all pots. Germinated seeds were transplanted 5 cm deep into the soil (6 liters of soil in pots with diameter of 25 cm).

The pots were divided such that twelve were for well-watered (WW) plants and 12 were for plants that were to be subjected to water deficit (WD). The transplanted seeds were allowed to grow by placing the pots in a greenhouse at the University of the Western Cape (-33.930917, 18.624778; i.e. 33°55'51.3"S 18°37'29.2"E); wherein the greenhouse was set to remain at a temperature of 29 °C for 16 hours (during the day) and a temperature of 18 °C during the night (8 hours dark period). Lighting during the day was supplemented with high pressure sodium lamps providing a photon flux density of 600 µmol m-² s^-1. Soil for WW plants was kept moist by irrigating the soil with 300 ml of tap water once a week (every Friday at midday) until the time of harvesting. The soil for WD plants did not receive water until the plants were harvested. WW and WD plants from both species were harvested 42 days after transplanting the germinated seeds into the soil, which was the time when reduction in leaf tissue turgor (apparent partial flaccidity of some of the leaves) was observed in some of the WD plants of both species.

2.2. Physiological and biochemical analyses

2.2.1. Soil water potential

The WP4C water potentiometer (Meter Group, Pullman, WA, USA) was used to measure soil water potential at the day of harvesting for the WW and the WD plants. Water potential measurement in the soil were done according to the instructions in the operating manual of the WP4C instrument in precise mode. Soil samples were selected randomly from WW and WD plants in triplicates for each Tylosema species for the measurements on the WP4C.

2.2.2. Leaf water potential

Leaf water potential was measured at midday on the day of harvesting in three of each of the WW and WD plants from each of the Tylosema species, using the Model 1505D Pressure Chamber Instrument (PMS Instruments, Albany, OR, USA), according to the instructions provided in the manual for the pressure chamber. For the leaf water potential, three biological replicates of the second youngest leaf from each treatment were harvested and used in the pressure chamber instrument.

2.2.3. Measurements of shoot and root weights

The WW and WD plants of each of the two Tylosema species were used to measure the fresh weights of roots and shoots of the plants by weighing them on a scale (Kern^® PLE 4200-2N precision scale, Kern and Sohn GmbH, Balingen, Germany).

2.2.4. Relative water content

The relative water content of the leaves of WW and WD T. esculentum and T. fassoglense (three biological replicates for each) was determined based on the formula:

$$RWC=100X{\displaystyle \frac{⌊FW-DW⌋}{⌊TW-DW⌋}}$$

; where RWC is the relative water content expressed as a percentage, FW is the fresh weight, TW is the turgid weight, and DW is the dry weight of the leaves from each treatment of each of the two species. To obtain FW, the second youngest fully open leaf of each plant was weighed on the Kern^® PLE 4200-2N precision scale, followed by weighing on the same scale after an overnight incubation in distilled H₂O (with excess surface water removed by dabbing with a paper towel) to obtain TW. The leaves were subsequently dried for 48 hours in a drying oven at 70 °C, immediately followed by weighing on the Kern^® PLE 4200-2N precision scale to obtain DW.

The remaining leaf material (the first, third, fourth, and fifth youngest leaves) and roots of each plant were harvested, ground in liquid nitrogen and stored as a fine powder at -80 °C until further use.

2.2.5. Proline content

Free proline content was measured in extracts from ground leaf material in three biological replicates. Each biological replicate was generated by combining leaf material from 4 plants of each species under each treatment. The procedure described by Ali et al. [30] was used for the spectrophotometry-based measurement of free proline.

2.2.6. Hydrogen peroxide and malondialdehyde contents

Hydrogen peroxide (H₂O₂) content was measured from extracts from the same leaf material used for the proline measurements. The procedure used for H₂O₂ measurements was performed as described by Ali and Ludidi [31]. The same extract used for H₂O₂ measurement was used to measure the malondialdehyde content in the leaves, which is indicative of the extent of leaf lipid peroxidation, as described by Ali and Ludidi [31].

2.2.7. Antioxidant enzyme activities

The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) enzymes were measured in protein extracts in triplicates using the spectrophotometric procedures described by Ali and Ludidi [31]. Each of the root samples used in these measurements were a pool from 4 plants from each species under each treatment. In these spectrophotometric measurements, the SOD assay was based on Beauchamp and Fridovich [32], APX activity was measured based on Nakano and Asada [33], the method followed for CAT activity was adapted from Lück [34], whereas the GR measurement was based on the method of Foyer and Halliwell [35].

Characterization of the responses of each of the antioxidant enzyme isoforms to water deficit was assessed based on changes in the intensity of bands in the images of gels obtained after native polyacrylamide gel electrophoresis (PAGE). The pixel intensities of the isoforms were calculated using AlphaEase 4.0 (Alpha Innotech Corporation, San Leonardo, CA). The native PAGE for SOD was conducted as described by Beauchamp and Fridovich [32], APX activity was assessed using the method of Seckin et al. [36], the occurrence of the CAT isoforms was detected based on Yamashita et al. [37], and GR isoforms were identified following the method of Rao et al. [38].

2.2.7. Statistical analysis

Results from each of the experiments described were analyzed using GraphPad Prism 6.0 (GraphPad Software, Boston, MA) using one-way analysis of variance (ANOVA), with the level of significance at 5% determined based on the built-in Tukey-Kramer test.

To explore the relationship between H₂O₂ contents, MDA levels and antioxidant enzyme activates in the two Tylosema species under water deficit stress, an R script was used to investigate the correlation between these parameters. The fold change of each parameter was calculated for both T. esculentum and T. fassoglense to determine the changes of these parameters within the two species. Subsequently, a fold change ratio for each parameter was computed by dividing the fold change of T. esculentum by that of T. fassoglense. The fold change ratios indicate how the studied parameters differ between the two species in response to water deficit stress. These fold change ratios were used to determine the correlation between the studied parameters using Pearson correlation coefficient.

3. Results

3.1. Plant water status and growth

Limitation of water supply to the soil mixture reduced soil water potential to equivalent levels in the soil in which the two Tylosema species were grown. At the time of harvesting, the soil water potential for the WW plants corresponded to approximately -0.24 MPa for T. esculentum and -0.26 MPa for T. fassoglense. However, for the WD plants, the soil water potential dropped to -0.83 and -0.81 MPa for T. esculentum and T. fassoglense plants, respectively (Figure 1A). Soil water deficit resulted in a reduction in the water potential of the leaves of both species, but the reduction in the leaf water potential in T. fassoglense was more prominent than in T. esculentum. The leaf water potential decreased from -0.66 to -1.1 MPa (1.6-fold) in response to water deficit in T. esculentum whereas it decreased from -0.65 to -1.5 MPa (2.3-fold) in T. fassoglense in response to water deficit (Figure 1B).

The decreased leaf water potential was accompanied by reduced shoot fresh weight in both Tylosema species (Figure 1C) and a reduced root fresh weight only for T. fassoglense (Figure 1D) in response to WD. The decrease in shoot fresh weight was more pronounced in T. fassoglense (1.5-fold, from 7.6 to 5.1 g/plant) than in T. esculentum (1.3-fold, from 4.8 to 3.7 g/plant) in response to water deficit (Figure 1C). Water deficit decreased root fresh weight by 1.5-fold (5.3 to 3.6 g/plant) in T. fassoglense, whereas no significant change was observed for T. esculentum root fresh weight between WD and WW conditions (Figure 1D).

WD reduced the relative water content from 96 % to 85 % (1.1-fold) in the leaves of T. esculentum, whereas the water deficit-induced decrease in T. fassoglense was from 96 % to 75 % (1.3-fold) (Figure 2A). These changes in leaf relative water content were accompanied by corresponding changes in leaf free proline content, in which a more pronounced increase in proline content occurred for T. fassoglense (3.3-fold, from 9.6 to 31.3 mmol.mg^-1 FW) than for T. esculentum (1.3-fold, from 9.4 to 12.3 mmol.mg^-1 FW) in response to WD (Figure 2B).

3.2. Oxidative stress and antioxidant responses

Water deficit (WD) did not significantly affect leaf hydrogen peroxide (H₂O₂) content in T. esculentum, whereas it caused an increase of 1.7-fold (299 to 518 nmol.g^-1 FW) in H₂O₂ content in the leaves of T. fassoglense (Figure 3A). The H₂O₂ response corresponded to similar changes in malondialdehyde (MDA) content, where no change was triggered by water deficit in T. esculentum but an increase of 1.2-fold (89.7 to 104 nmol.g^-1 FW) occurred in T. fassoglense in response to WD (Figure 3B).

Superoxide dismutase (SOD) activity increased by 1.5-fold (1.5 to 2.3 units.mg^-1 protein) in the leaves of T. esculentum, while only 1.2-fold increase (2.5 to 3.1 units.mg^-1 protein) was observed in T. fassoglense in response to WD (Figure 4A). For roots, WD increased the superoxide dismutase activity in T. esculentum by 4.5-fold (0.3 to 1.1 units.mg^-1 protein) but had no significant effect on the superoxide dismutase activity of T. fassoglense roots (Figure 4B). Leaf tissue showed a total of six superoxide dismutase isoforms in both T. esculentum and T. fassoglense, with noticeable increase in the intensities of SOD 2 (1.3-fold, from 7.3 to 9.3 arbitrary units) and SOD 3 (1.3-fold, 7.7 to 10.3 arbitrary units) in response to WD in T. esculentum, whereas a noticeable increase for SOD 1 (1.7-fold, from 6.3 to 10.7 arbitrary units), SOD 2 (1.2-fold, from 9.3 to 11.3 arbitrary units) and SOD 6 (1.2-fold, from 9.3 to 10.7 arbitrary units) were observed for T. fassoglense (Figure 4C, Supplementary Table 1). Roots of both species showed a total of two SOD isoforms, namely SOD 3 and SOD 4 (Figure 4C, Supplementary Table 1). No SOD isoforms were visible for T. esculentum under well-watered conditions in roots, but WD induced the appearance of SOD 3 by 2.2-fold (5.7 to 12.3 arbitrary units) and SOD 4 by 1.4-fold (5.7 to 7.7 arbitrary units) in the roots of this species (Figure 4C, Supplementary Table 1). No clear change in the superoxide dismutase isoforms occurred in roots of T. fassoglense in response to WD (Figure 4C, Supplementary Table 1).

Catalase (CAT) activity increased in the leaves and roots of both species, whereby the leaf CAT activity for T. esculentum increased by 2-fold (75 to 150 µmol.min^-1.mg^-1 protein) and that for T. fassoglense increased by 2.7-fold (45 to 102 µmol.min^-1.mg^-1 protein) (Figure 5A), whereas the root CAT activity showed an increase of 1.8-fold (6.7 to 12.2 µmol.min^-1.mg^-1 protein) and 2.6-fold (3.5 to 8.6 µmol.min^-1.mg^-1 protein) in T. esculentum and T. fassoglense, respectively (Figure 5B). Two CAT isoforms were observed in the leaves of the two Tylosema species, wherein the water deficit increased CAT 1 and CAT 2 in both species (Figure 5C, Supplementary Table 2). T. fassoglense exhibited a higher intensity increase for these isoforms, with a 1.5-fold increase (41 to 61 arbitrary units) for CAT 1, and an increase of 1.4-fold (71 to 97.7 arbitrary units) for CAT 2. In contrast, T. esculentum, showed an increase of 1.3-fold (68.3 to 86.3 arbitrary units) for CAT 1 intensity and a 1.2-fold increase for CAT 2 (90.3 to 111 arbitrary units) intensity (Figure 5C, Supplementary Table 2). Three CAT isoforms were detected in the roots of the two species, with T. esculentum showing enhanced intensity of 1.3-fold for CAT 2 (6.3 to 8.3 arbitrary units) and 1.7-fold for CAT 3 (5.3 to 9.0 arbitrary units) in response to WD, while T. fassoglense showed an increased intensity only for CAT 2 (1.4-fold, from 5.7 to 7.7 arbitrary units). Root CAT activity was notably lower than leaf CAT activity, wherein CAT 3 and CAT 2 were the most prominent root isoforms in T. esculentum and T. fassoglense, respectively (Figure 5C, Supplementary Table 2).

Leaf ascorbate peroxidase (APX) activity exhibited a 1.2-fold increase (2.5 to 3.0 µmol.min^-1.mg^-1 protein) in T. esculentum and a 1.5-fold increase (2.4 to 3.6 µmol.min^-1.mg^-1 protein) in T. fassoglense (Figure 6A), whereas the increase was not significant in either T. esculentum or T. fassoglense roots (Figure 6B). Five APX isoforms (APX 1, 2, 3, 4, and 5) were observed in the leaves and roots of the two Tylosema species, with the most prominent isoform being APX 5, as the intensity of the rest of the isoforms was low, although APX 1 was also prominent in the roots of both species (Figure 6C, Supplementary Table 3).

The assessment of glutathione reductase (GR) activity revealed that WD augmented leaf GR activity only in T. fassoglense from 15 to 21 nmol.min^-1.mg^-1 protein (1.4-fold), but not in T. esculentum (Figure 7A). However, GR activity was increased in response to WD in the T. esculentum roots by 1.3-fold (34 to 45 nmol.min^-1.mg^-1 protein) but decreased by 1.7-fold (44 to 26 nmol.min-1.mg-1 protein) in the roots of T. fassoglense (Figure 7B). Five isoforms of GR (GR 1, 3, 5, 6, and 7) were observed in the leaves of the two Tylosema species and six (GR 1, 2, 3, 4, 5, and 6) were observed in their roots (Figure 7C). In the leaves, only the intensity (and thus activity) of GR 7 was increased in T. esculentum (1.2-fold, from 6.0 to 7.3 arbitrary units) in response to WD (Figure 7C, Supplementary Table 4). In T. fassoglense leaves, only GR 1 and GR 5 activities were up-regulated in response to WD, with an increase of 1.4-fold (6.7 to 9.7 arbitrary units) for GR1 and an increase of 1.3-fold (6.7 to 8.7 arbitrary units) for GR 5, whereas the rest of the isoforms were not affected (Figure 7C, Supplementary Table 4). GR 1, GR 2, GR 3, and GR 4 were increased by 1.2-fold, 1.6-fold, 1.3 and 1.4-fold (21 to 26, 14 to 23, 8.3 to 11 and 7.7 to 11, arbitrary units), respectively in T. esculentum roots in response to WD. The intensities of all GR isoforms were down-regulated in T. fassoglense roots, except for the T. fassoglense GR 5 isoform, which was induced by 1.3-fold (6.0 to 8.0 arbitrary units) under WD (Figure 7C, Supplementary Table 4).

Pearson correlation analyses revealed relationships among various biochemical responses to water deficit in the two Tylosema species, as illustrated in Figure 8. A moderate positive correlation was observed between leaf H₂O₂ and MDA contents. H₂O₂ exhibited a moderate negative correlation with SOD activity in both leaf and root tissues. A strong negative correlation was observed between H₂O₂ and CAT activity in both leaves and roots. Notably, H₂O₂ exhibited a perfect negative correlation with APX activity in the leaves and a strong positive correlation with root GR activity.

4. Discussion

Members of the genus Tylosema are known for their exceptional drought tolerance, but the mechanisms of their adaptation to drought vary and appear to depend on the specific species and their natural habitat [16]. Several physiological responses of Tylosema esculentum to drought, high temperature and high radiation were reported by Mitchell et al. [11], and by Karamanos and Travlos [39]. However, very little is known about the comparative responses between T. esculentum and T. fassoglense under similar water deficit (WD) stress. In this study, we exposed T. esculentum and T. fassoglense to well-watered (WW) and WD treatments under greenhouse conditions. Following the treatment period, we observed no significant differences in the soil water potential of the WW soil in which T. esculentum and T. fassoglense were grown (approximately -0.25 MPa). When we imposed WD conditions, the soil water potential dropped to approximately -0.8 MPa for the treated Tylosema species. This result demonstrated that the two Tylosema species experienced the same level of soil WD conditions at the time of harvesting.

Tylosema plants exhibit adaptation to reduce transpiration-induced water loss through early stomatal closure [11]. In this study, we measured the leaf water potential of the two species under contrasting water regimes. The two species did not show any differences in leaf water potential when grown under WW conditions. However, both species showed reductions in leaf water potential when exposed to WD conditions. The reduction in leaf water potential was even higher in T. fassoglense than in T. esculentum. We thus conclude that T. esculentum maintains leaf water status better than T. fassoglense when both plants are exposed to soil WD. Karamanos and Travlos [39] observed similar leaf water potential levels when T. esculentum, collected from Botswana, were exposed to WD stress under greenhouse conditions. Karamanos and Travlos [39] concluded that leaf behavior plays a vital role in the regulation of the water status in T. esculentum under WD stress. The observed changes in leaf relative water content in the two Tylosema species indicate that T. esculentum has better leaf water retention capacity than T. fassoglense.

Maintaining a larger root biomass under drought stress is part of the adaptive responses of plants to cope with limited water availability [40]. Our results show that T. esculentum underwent less leaf biomass reduction than T. fassoglense, and that WD stress did not alter the root fresh weight of T. esculentum, whereas significant root weight reductions occurred in T. fassoglense. This suggests that the lower reduction in shoot biomass observed in T. esculentum could be attributed to its ability to maintain root biomass under WD stress. It was established that drought-tolerant plants can maintain their root length and root volume to improve their capacity to take up the limited water in the soil [41].

Proline often accumulates in plants under drought stress, wherein it functions as an osmoprotectant and ROS scavenger [42]. In our study, T. fassoglense leaves accumulated more proline than the T. esculentum leaves, which could indicate that the T. fassoglense leaves experienced higher WD stress levels than T. esculentum leaves. The higher accumulation of proline observed in T. fassoglense did not assist the plant to maintain leaf biomass, which suggests that differences in WD stress tolerance between these two species is not associated proline content. Lv et al. [43] reported similar findings where no correlation between proline content and stress tolerance was observed in 25 rice cultivars.

Hydrogen peroxide (H₂O₂) accumulates to high levels in drought-exposed plants and leads to overaccumulation of malonaldehyde (MDA), resulting in lipid membrane damage [44]. In our study, we observed no changes in H₂O₂ and MDA contents in T. esculentum leaves exposed to WD stress. However, we observed a significant increase in H₂O₂ and MDA in the leaves of T. fassoglense exposed to WD. We suggest that the increase in leaf H₂O₂ led to more membrane damage, as evidenced by the increased MDA levels in T. fassoglense under WD stress than in T. esculentum. From this result, we conclude that T. esculentum limits ROS accumulation better under WD than T. fassoglense, which leads to better WD stress tolerance in T. esculentum than in T. fassoglense. Keyster et al. [45] demonstrated that the capacity to control ROS content is a key determinant of sensitivity and tolerance of plants to stress. In this study, we measured various ROS-antioxidant enzymes in order to further understand the differential ROS and MDA accumulation in T. esculentum and T. fassoglense under WD. We measured the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), as both total enzymatic activity and isoform analysis by native-PAGE staining.

SOD regulates the dismutation of superoxide radicals into H₂O₂ in different plant organelles [46]. Therefore, SOD is a key enzyme in the regulation of intracellular ROS levels and maintains redox homeostasis under oxidative stress caused by drought [47]. In this study, in response to WD, both leaf and root total SOD enzymatic activity was increased in T. esculentum, while no change was observed in the root total SOD enzymatic activity of T. fassoglense, which indicates that T. fassoglense does not have sufficient SOD capacity in the roots to control excessive ROS accumulation under WD condition. Isoform analysis indicated that SOD 2 and SOD 3 contribute the most to the total SOD increase in T. esculentum leaves under WD, whereas SOD 1, SOD 2 and SOD 6 account for the total SOD increase in T. fassoglense leaves. Furthermore, in T. esculentum roots, the increase in total SOD activity is mostly regulated by SOD 3 and SOD 4. One the other hand, these isoforms remained unchanged in T. fassoglense roots. This implies that the regulation of SOD isoform activity differs between T. esculentum and T. fassoglense under WD. Huseynova et al. [48] concluded that the differential regulation of a Mn-SOD in wheat cultivars contributed to differential sensitivity to drought stress. Wang et al. [49] observed similar results when exposing two rice cultivars to drought stress conditions where the drought-tolerant cultivar had a higher SOD activity than the drought-sensitive one. This observation was also reported by Singh et al. [50], who showed that drought-tolerant cotton cultivars had a higher SOD activity than cotton cultivars that are sensitive to drought stress.

CAT and APX have contrasting affinities for H₂O₂, indicating the existence of complementary metabolic roles of these two antioxidant enzymes in plants [51]. Corpas [52] proposed that APX may be an important enzyme for fine-tuning and scavenging low concentrations of H₂O₂, whereas CAT seems to be a key scavenger enzyme when H₂O₂ concentrations are high. Our results showed that although WD led to a higher increase in leaf and root total CAT enzymatic activity in T. fassoglense than in T. esculentum, the overall activity was higher in T. esculentum under both water conditions. This suggests that T. esculentum possesses a higher basal capacity for oxidative stress management, potentially contributing to its better WD tolerance. In addition, we observed a more pronounced increase in the CAT 2 and CAT 3 isoforms in T. esculentum compared to T. fassoglense leaves under WD. Therefore, our results suggest that these two CAT isoforms are important in regulating the level of H₂O₂; and the capacity of T. esculentum in increasing the activity of these isoforms under WD conditions is likely to contribute to its drought tolerance. These two isoforms may have distinct substrate affinities or subcellular localizations, allowing them to efficiently scavenge H₂O₂, highlighting the complex interplay between total antioxidant enzyme activity, isoform-specific activity and ROS regulation. Our APX data showed a higher increase in total leaf APX activity in T. fassoglense compared to T. esculentum. However, we noticed no significant difference in the total root APX activity of both species in response to WD. We hypothesize that leaf APX activity is much more important than root APX activity in H₂O₂ regulation under WD stress in Tylosema species. We also suggest that the regulation of APX 5 activity is important for WD responses because T. esculentum experienced a much lower decrease in APX 5 activity in roots under WD stress, which could also be correlated to its better H₂O₂ detoxification capacity. These results suggest that H₂O₂ scavenging capacity differs in the two Tylosema species under WD stress. Chugh et al. [53] observed that differences in CAT and APX activities correlated with the tolerance or sensitivity to drought stress in maize cultivars.

GR maintains the intracellular glutathione (GSH) pool in the reduced state [54]. GSH can function as a direct antioxidant (non-enzymatically) or an indirect antioxidant by recycling ascorbic acid [55]. Ahmad et al. [56] concluded that differential regulation of sulfur metabolism by GR in leaves and roots was associated with enhanced drought stress response in maize. In our study, total GR activity was increased in the leaves of T. fassoglense but no significant change was observed in T. esculentum in response to WD stress. Contrastingly, we observed an increase in the root GR activity in T. esculentum, while a decrease in GR activity was observed in the roots of T. fassoglense in response to WD stress. The GR isoform analysis indicated that GR 1 and GR 5 are the isoforms likely responsible for the increased GR activity in the leaves of T. fassoglense in response to WD. Furthermore, isoforms GR 1, GR 2, GR 3, and GR 4 may be responsible for the increase in the GR activity of T. esculentum roots in response to WD. We suggest that the increased GR activity in the roots of T. esculentum possibly contributed to its greater maintenance of root biomass and oxidative stress tolerance than T. fassoglense. Similar findings were reported by Khanna et al. [57]. These authors observed an increase in GR activity in the roots of a tolerant maize genotype in response to heat stress and a decrease in the activity in the leaves, while no significant change in GR activity was observed in both leaves and roots of the sensitive genotype. Zhang and Kirkham [58] observed that sorghum plants maintained a much higher increase in GR activity under drought stress when compared to sunflower.

The correlation coefficients provided insights into how the studied parameters are interconnected, contributing to a deeper understanding of the adaptive responses in the two Tylosema species to water deficit stress. The observed positive correlation between H₂O₂ and MDA contents suggests that the increase in H₂O₂ contributed to the increase in MDA content observed in the leaves of the two Tylosema species under water deficit stress. The negative correlation observed between H₂O₂ and CAT activity in both leaf and root tissues demonstrates the significance of CAT in mitigating excessive H₂O₂ levels in Tylosema species under water deficit. The perfect negative correlation of leaf APX activity with H₂O₂ further highlights the crucial role of leaf APX in the regulation of H₂O₂ in Tylosema species under water deficit stress. The positive correlation observed between GR activity in the roots and H₂O₂ contents could be linked to its key role in oxidative stress regulation via the regeneration of reduced glutathione (GSH), which gets oxidized by several pathways linked to H₂O₂ scavenging [59]. This correlation highlights the key role of GR activity in drought responses of Tylosema species and further indicates that the observed increase in GR activity in the roots of T. esculentum potentially contributes to its better efficiency in H₂O₂ detoxification than T. fassoglense.

5. Conclusions

This comparative study revealed that T. esculentum maintains greater water retention compared to T. fassoglense under the water deficit. Accumulation of proline under WD was not translated into enhanced water status in T. fassoglense, suggesting alternative adaptive mechanisms could govern water relations in these two Tylosema species. However, the higher water retention in T. esculentum could be attributed to its ability to maintain root biomass under WD. Furthermore, this study highlights the critical role of antioxidant enzyme activities in mediating tolerance to WD stress in the Tylosema species. Our findings indicate that T. esculentum demonstrated a higher SOD activity, which led to an effective control of ROS levels. The higher baseline of CAT total activity in both leaf and root tissues of T. esculentum plays an important role in its greater ability of oxidative stress management. The decrease in APX 5 activity was much lower in T. esculentum, indicating its potential role in mediating drought responses in the two species. Moreover, GR activity also exhibited differing patterns between the two species, with T. fassoglense showing an increase in GR activity in leaves but a decrease in roots, while T. esculentum displayed increased GR activity in the roots. This differing GR activity was associated with different levels of H₂O₂ detoxification, indicating that upregulation of GR activity, particularly in the roots, plays a key role against WD-induced oxidative damage in T. esculentum. These findings emphasize the complexity of the antioxidant defense system, suggesting that more finely tuned regulatory mechanisms may play a significant role in ROS detoxification under WD stress.

The present work offers a foundation for further research in elucidating the genetic basis of drought adaptation in the genus Tylosema. Understanding the molecular adaptive mechanisms underlying the extreme drought tolerance in T. esculentum and how they differ in T. fassoglense could offer potential avenues for enhancing drought tolerance in T. fassoglense and other leguminous crops. This enhancement of crop tolerance to drought is essential for sustainable agriculture and global food security.