Exogenous alpha-lipoic acid treatments reduce the oxidative damage caused by drought stress in two grapevine rootstocks

2.1 Plant material and growing conditions

The investigation was conducted in 2022 at the research greenhouse located on the premises of Yozgat Bozok University's Faculty of Agriculture, which is situated at an elevation of 1340 meters in Yozgat, Turkey (coordinates: 39°46′ N, 34°48′ E). Specimen measurements and analyses were performed in the university's research laboratories. The plant material, consisting of rootless cuttings, was sourced from a collection vineyard affiliated with the Agricultural Research Institute in the Transitional Zone of the Middle Black Sea, located in Tokat, Turkey. In this study, two American grapevine rootstocks were utilized: the drought-sensitive Vitis riparia Michaux × Vitis rupestris Scheele ‘3309 Couderc' (3309 C) and the drought-tolerant Vitis berlandieri Planchon × Vitis rupestris Scheele hybrid ‘1103 Paulsen’ (1103 P). These selections were based on their documented responsiveness to drought conditions in previous research. The young grapevine plants were positioned in containers measuring 11 × 11 × 22 cm, each filled with a sterile mix of peat and perlite in a 1:1 ratio. Following planting, the growth medium was fertilized with a full-strength nutrient solution (pH adjusted to 6.5), as recommended by Ollat et al. (1998), tailored for the nourishment of young grapevine plants All plants were irrigated to 100% field capacity (FC) until the start of the drought trial. The greenhouse environment was regulated to maintain a relative humidity of approximately 70 ± 2%, a temperature of 25 ± 5°C, and exposure to natural light. This climate was facilitated by a combination of heating systems, fans, and a pad system, which collectively supported the optimal development of the plants' root and shoot systems. Environmental conditions within the greenhouse were closely monitored using a digital thermohygrometer (ISOLAB) located centrally. During the initial growth phase, each sapling was allowed to develop a single shoot following bud break from single-bud cuttings. Eight weeks post-planting, upon the satisfactory development of both root and shoot systems, saplings that displayed consistent leaf area and stem diameter were carefully selected.

2.2 Treatments and experimental design

The weight and moisture content of the pots were monitored daily and watered daily with a full-strength nutrient solution (Ollat et al., 1998) until harvest to maintain 20 and 60% FC humidity of the growing medium throughout the entire experimental period. A 1:1 mixture of peat:perlite was added in pots with a volume of approximately 2.5 L. Considering the volume occupied by the plant roots and the space left at the top of the pots, the volume of the growing medium was about 2 L. Therefore, each pot contained 1 L of peat and 1 L of perlite. However, these growing media were not compacted too much to ensure the air balance needed for the plant roots. The growing medium was, therefore, of medium density. The amount of water required for the growing medium to reach 100% field capacity was initially 1.3 L. All plants were watered at this rate at the beginning of the experiment. Until the onset of drought stress treatments (for eight weeks), all plants were irrigated daily with about 100 mL of water to recover water lost through daily plant water consumption + daily evaporation (about 7.5%). When drought stress treatments were initiated, the amount of water given to the plants daily was 60 mL to maintain 60% FC and 20 mL to maintain 20% FC. This rate increased to 75 mL for 60% FC and 35 mL for 20% FC towards the end of the experiment (especially between the 35 and 40th days) due to plant growth and increased water consumption in parallel. The investigation of the impact of α-Lipo on grapevine saplings under drought conditions initiated on the ninth day of the drought stress intervention (Yang et al., 2023). This timing was chosen to support natural defence mechanisms, reduce oxidative damage and increase plant resistance in both rootstocks. An initial stock solution of α-lipoic acid (Sigma Aldrich, St. Louis, MO, USA, CAS: 1077-28-7) was prepared at a concentration of 1 mM using distilled water. This solution was then diluted to achieve various concentrations for the study, establishing a treatment range of 1–50 μM of α-Lipo. Control groups were treated with distilled water. For the experiment, Tween-20 (0.05%, v/v, Sigma Aldrich, CAS: 9005-64-5) served as a non-ionic surfactant. Solutions prepared at different concentrations for the treatments were transferred into 1-L spray bottles and sprayed on the leaves of the saplings at the time of application. The treatments were applied at 5:30 p.m. to completely wet all green parts of the grapevine saplings, as detailed by Ramadan et al. (2022). Control sapling leaves without α-Lipo treatment received a 25 mL/grapevine sapling solution of distilled water and 0.05% (v/v) Tween-20. α-Lipo-treated saplings were sprayed with solutions of 1, 25, and 50 μM α-Lipo combined with Tween-20 (0.05%, v/v) at 25 mL/grapevine sapling. Treatments were repeated for seven consecutive days. The experiment was comprised of eight treatments:

The experiment was arranged as a completely randomized design with three subsamples per plot and six replications. In total, 180 pots were employed, covering both American grapevine rootstocks across five treatments. Saplings were allowed to grow for three weeks after the last foliar treatment application before data collection was initiated. Subsequently, sampling was conducted to evaluate growth characteristics and other stress-related parameters. The experimental period spanned from May 1, 2022, to August 15, 2022, after which the experiment concluded. Some morphological, physiological, and biochemical assessments were carried out to determine the effects of α-Lipo treatments on alleviating drought stress impacts on the saplings. Harvesting involved selecting the fourth leaf from the apex of each shoot from the grapevine saplings. Roots were isolated from the peat and perlite mixture and rinsed with deionized water. For biochemical analyses, leaf samples were collected immediately following the 40-day drought stress regimen, rapidly frozen in liquid nitrogen, and stored at −80°C in an ultra-deep freezer (Forma-89000 Series, Thermo Scientific) until analysis (Aazami and Mahna, 2017).

2.3 Evaluation of some morphological parameters

Shoot and root lengths were assessed using a standard ruler. The determination of shoot and root fresh weights involved the utilization of a digital balance (AXIS, AGN200C), while the measurement of shoot and root dry weights ensued after thorough desiccation in a ventilated oven at 105°C. The number of leaves was determined by counting all leaves from the tip of the shoot to the base, taking the first fully opened leaf as the first leaf. Leaf thickness was measured using a mechanical micrometer (BTS-12051, BTS Mechanics). The average leaf area was measured using an Area Meter (AM 300, ADC BioScientific Ltd.), a device specifically designed for this purpose. The upper fourth leaves of the shoots were used for leaf area and leaf thickness measurements.

2.4 Evaluation of some physiological, and biochemical parameters

For all physiological and biochemical measurements, the upper fourth leaves of the shoots were used. Chlorophyll content was measured between the main veins of the leaves using a portable chlorophyll meter (SPAD-502, Konica Minolta). Stomatal conductance and leaf temperature were measured using a leaf porometer (Decagon, SC-1, Pullman) during the period of midday sunlight, specifically from 11:00 a.m. to 01:00 p.m. Relative water content (RWC) was computed following the methodology outlined by Abd El-Gawad et al. (2021). Proline levels were determined spectrophotometrically (Lambda 25, Perkin Elmer, Inc., USA) at 520 nm employing the ninhydrin assay, following the procedure outlined by Bates et al. (1973). The degree of membrane damage was calculated following the method outlined by Lutts et al. (1996), involving the measurement of electrolyte release from the cell. Lipid peroxidation levels were assessed by quantifying the quantity of malondialdehyde (MDA) generated through the thiobarbituric acid reaction, following the methodology outlined by Heath and Packer (1968). Extracts were prepared following the methodology elucidated by Özden et al. (2009). The specific activity of superoxide dismutase (SOD), Catalase (CAT), and ascorbate peroxidase (APX) enzymes was calculated after determining the total soluble protein in the supernatant, following the procedure described by Bradford (1976). The evaluation of SOD (EC 1.15.1.1) functionality involved gauging its effectiveness in impeding the photochemical reduction process of nitro blue tetrazolium at the wavelength of 560 nm (Agarwal and Pandey, 2004). The CAT (EC 1.11.1.6) functionality was established by observing the reduction in absorbance of H₂O₂ at 240 nm, as outlined in Gong et al. (2001). The assessment of APX (EC 1.11.1.11) functionality relied on observing the decline in ascorbate levels at a wavelength of 290 nm (Nakano and Asada, 1981).

2.5 Data analysis

Data were recorded using the Microsoft 365 Excel program. The quantitative dataset, encapsulating the responses of grapevine rootstocks (3309 C and 1103 P) to varying concentrations of α-Lipo under well-irrigated and drought stress conditions were subjected to a comprehensive variance analysis (Three-Way ANOVA) utilizing IBM SPSS Statistics V22.0 software. This analysis detected the variances attributed to the principal treatments (rootstock, α-Lipo and irrigation) and their interactive effects. Attributes lacking interaction effects were further analysed through One-Way ANOVA to assess within-group variances. The significance of variations among treatment means was determined using Duncan's multiple range test, with a significance level established at p ≤ 0.05. Results were presented as mean values accompanied by standard deviations (SD), offering a clear depiction of the data's distribution and variability. Furthermore, a principal component analysis (PCA) was performed using GraphPad Prism version 9.3.1 (GraphPad Software, LLC) to visualize the direction and strength of the relationship between the studied traits and the results were described using a biplot following the methodology outlined by Evgenidis et al. (2011). In addition, a hierarchical clustering heatmap was created using the SRPLOT online platform (https://www.bioinformatics.com.cn/en accessed 31 May 2024) to visualize and relate the relationships and densities between the factors and the studied traits.

3.1 The effect of α-lipoic acid application on some morphological parameters in rootstocks under drought stress

Different concentrations of α-Lipo treatments caused significant changes in growth parameters of 3309 C and 1103 P rootstocks under well-irrigated and drought conditions. In 1103 P under well-irrigated conditions, α-Lipo application, especially at a concentration of 1 μM, significantly increased shoot length and provided the highest average of 49.97 cm (Figure 2A, Table S3). For the same rootstock, 25 and 50 μM α-Lipo treatments also increased shoot length (47.05 and 47.28 cm, respectively), but with lower effects compared to 1 μM treatment. At 3309 C, a different trend was observed, with the highest effects of 40.12 and 39.52 cm obtained from 25 and 50 μM α-Lipo treatments in well-irrigated conditions, respectively, while the effect of 1 μM α-Lipo in the same conditions was lower (37.21 cm). These results indicate that α-Lipo concentration and irrigation status may have different effects depending on the rootstock type, and that 1103 P showed the best performance, especially under well-irrigated and 1 μM α-Lipo applied conditions, while 3309 C provided the highest values under well-irrigated and α-Lipo applied conditions in the range of 25–50 μM. Both rootstocks under drought stress had lower shoot length averages when α-Lipo was not applied. 3309 C rootstock under drought stress and without α-Lipo treatment showed the lowest values in terms of shoot length with 14.00 cm. This value was followed by 1103 P rootstock under the same conditions with 17.50 cm. Under both well-irrigated conditions and drought stress, 1103 P had a longer shoot compared to 3309 C when α-Lipo was not applied. A similar situation occurred at different α-Lipo concentrations under well-irrigated conditions. On the other hand, the difference between 1103 P and 3309 C rootstocks was insignificant after 1 and 25 μM α-Lipo treatments under drought conditions. At 1103 P, all α-Lipo concentrations in well-irrigated conditions significantly increased shoot fresh weight, with the highest means of 18.44, 18.94 and 17.21 g for 1, 25 and 50 μM, respectively (Figure 2B, Table S3). However, these values were in the same statistical group with the values of 16.94 and 17.22 g, respectively, obtained from 25 and 50 μM α-Lipo treatments under well-irrigated conditions, which showed the highest effects at 3309 C. In contrast, the effect of 1 μM α-Lipo in well-irrigated conditions was lower at 3309 C (13.38 g). These results indicate that α-Lipo concentration and irrigation status may have different effects on shoot fresh weight values depending on the rootstock type and that 1103 P generally exhibited high performance at all α-Lipo concentrations under well-irrigated conditions, while 3309 C provided the highest values under well-irrigated conditions and α-Lipo applied in the range of 25–50 μM. Both rootstocks under drought stress showed lower shoot fresh weight averages in the absence of α-Lipo or in the presence of low concentrations of α-Lipo. Drought-stressed rootstock 3309 C with 0 and 1 μM α-Lipo exhibited the lowest values for shoot fresh weight with 2.70 and 3.14 g, respectively. These averages were in the same statistical group with the values of 3.00 and 4.03 g obtained from 1103 P under the same conditions. The application of 1 μM α-Lipo under well-irrigated conditions and 25 μM α-Lipo under drought stress resulted in higher shoot fresh weight values of 1103 P compared to 3309 C. In contrast, other α-Lipo concentrations under both well-irrigated and drought-stress conditions did not cause any significant difference between 1103 P and 3309 C rootstocks. In terms of shoot dry weight, the highest averages of 3.00, 3.24 and 3.17 g were obtained from 0, 25 and 50 μM α-Lipo treatments of 1103 P under well-irrigated conditions, respectively (Figure 2C, Table S3). However, these values were in the same statistical group with 2.89, 3.03 and 2.95 g obtained from 1, 25 and 50 μM α-Lipo concentrations at 3309 C under well-irrigated conditions, respectively. Therefore, α-Lipo treatments in well-irrigated conditions did not cause a significant change in shoot dry weight values of 1103 P compared to the control without α-Lipo, whereas all α-Lipo concentrations applied at 3309 C in the same conditions resulted in a significant increase in shoot dry weight values compared to the control without α-Lipo. These results indicated that α-Lipo concentration and irrigation status may have different effects on shoot dry weight values depending on the rootstock type, and 1103 P exhibited higher averages than 3309 C under well-irrigated conditions without α-Lipo treatment, while it had similar averages with 3309 C after α-Lipo treatments. In both rootstocks under drought stress, the lowest shoot dry weight averages were determined under non-α-Lipo conditions. These values were 0.70 and 0.50 g for 1103 P and 3309 C rootstocks, respectively. Under drought stress, 1 μM α-Lipo treatment resulted in higher shoot dry weight values for 1103 P compared to 3309 C. In contrast, other α-Lipo concentrations under drought stress conditions did not cause any significant difference between 1103 P and 3309 C rootstocks.

In 1103 P under well-irrigated conditions, α-Lipo application, especially at 1 μM concentration, significantly increased root length and obtained the highest average of 43.48 cm (Figure 2D, Table S4). For the same rootstock, 25 and 50 μM α-Lipo treatments also increased root length (37.07 and 38.19 cm, respectively), but with lower effects compared to 1 μM treatment. At 3309 C, a different trend was observed and the highest effects of 25 μM and then 50 μM α-Lipo treatments in well-irrigated conditions were 38.92 and 35.34 cm, respectively, while the effect of 1 μM α-Lipo in the same conditions was lower (27.46 cm). These results indicate that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type, and that 1103 P showed the best performance, especially in well-irrigated and 1 μM α-Lipo treated conditions, while 3309 C provided the highest values in well-irrigated and 25 μM α-Lipo treated conditions. Both rootstocks under drought stress had lower root length averages when α-Lipo was not applied. 3309 C rootstock under drought stress and without α-Lipo treatment showed the lowest value in root length with 14.50 cm. This value was in the same statistical group with 16.17 cm obtained from 1103 P rootstock under the same conditions. Under well-irrigated conditions, 1103 P had longer roots compared to 3309 C in the presence (except for 25 μM α-Lipo application) and absence of α-Lipo. In contrast, the difference in root length between rootstocks 1103 P and 3309 C was insignificant at all α-Lipo concentrations applied under drought conditions. In 1103 P, α-Lipo applications at concentrations of 1 and 25 μM under well-irrigated conditions significantly increased root fresh weight and provided the highest means of 14.17 and 14.05 g, respectively (Figure 2E, Table S4). For the same rootstock, 50 μM α-Lipo treatment also increased root fresh weight (12.29 g), but with a lower effect compared to 1 and 25 μM α-Lipo treatments. At 3309 C, the highest effects were obtained from 25 μM and 50 μM α-Lipo treatments in well-irrigated conditions (12.68 and 11.89 g, respectively), while the effect of 1 μM α-Lipo in the same conditions was lower (8.94 g). These results indicate that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type and that 1103 P performed best, especially under well-irrigated conditions and low or medium concentrations of α-Lipo, while 3309 C achieved the highest values under well-irrigated conditions and medium or high concentrations of α-Lipo. In both rootstocks under drought stress, lower root fresh weight averages were found in the non-α-Lipo treated groups. Accordingly, 3309 C rootstock, which was under drought stress and not treated with α-Lipo, exhibited the lowest value in terms of root fresh weight with 1.59 g. This value was in the same statistical group with 2.23 g obtained from 1103 P rootstock under the same conditions. Under well-irrigated conditions, 1103 P had higher root fresh weight averages compared to 3309 C in the presence (except 50 μM α-Lipo application) and absence of α-Lipo. In contrast, under drought conditions, the difference between root fresh weight of 1103 P and 3309 C rootstocks was insignificant at all α-Lipo concentrations applied.

The highest averages for root dry weight values were 2.79 and 2.63 g for 1 μM α-Lipo concentration of 1103 P under well-irrigated conditions and 25 μM α-Lipo concentration of 3309 C under the same conditions, respectively (Figure 2F, Table S4). For 1103 P, 25 and 50 μM α-Lipo treatments also increased root dry weight (2.47 and 2.53 g, respectively), but with lower effects compared to 1 μM treatment. In 3309 C, unlike 1103 P, the highest effects of 2.63 and 2.43 g were obtained from 25 and 50 μM α-Lipo applications in well-irrigated conditions, while the effect of 1 μM α-Lipo in the same conditions was lower (1.91 g). These results indicate that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type and that 1103 P showed the best performance, especially under well-irrigated and 1 μM α-Lipo applied conditions, while 3309 C provided the highest averages in terms of root dry weight values under well-irrigated and α-Lipo applied conditions in the range of 25–50 μM. Both rootstocks under drought stress had the lowest root dry weight averages when α-Lipo was not applied (1103 P: 0.93 g; 3309 C: 0.82 g). These averages were statistically in the same group with the 1 μM α-Lipo concentration of 3309 C under drought stress (1.04 g). The difference between the root dry weight values of 1103 P and 3309 C rootstocks as a result of different concentrations of α-Lipo treatments under both well-irrigated conditions and drought stress (except 25 μM α-Lipo treatment under well-irrigated conditions) was found to be insignificant. The highest values in terms of the number of leaves were obtained from all α-Lipo concentrations of 1103 P under well-irrigated conditions (13.55–14.07 pcs) and 3309 C under the same conditions with 25 and 50 μM α-Lipo concentrations of 13.64 and 13.55 pcs, respectively (Figure 3A, Table S5). Unlike 1103 P, 3309 C showed a lower number of leaves value (12.52 pcs) under 1 μM α-Lipo conditions. These results suggest that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type and that 1103 P performed better in terms of the number of leaves value, especially under well-irrigated and 1 μM α-Lipo applied conditions, while 3309 C under the same conditions provided lower values. Both rootstocks under drought stress had the lowest number of leaves averages when α-Lipo was not applied. Rootstock 3309 C under drought stress conditions and without α-Lipo treatment showed the lowest values in terms of the number of leaves with 6.00 pcs. This value was followed by 1103 P rootstock under the same conditions with 6.67 pcs. Under drought stress, 1103 P had higher number of leaves than 3309 C at 0, 25 and 50 μM α-Lipo concentrations. A similar situation was observed at 1 μM α-Lipo concentration under well-irrigated conditions. The highest values for leaf thickness were 0.35 mm at 50 μM α-Lipo concentration in 1103 P under drought conditions (Figure 3B, Table S5). In 3309 C under the same conditions, 25 and 50 μM α-Lipo concentrations provided higher values compared to the α-Lipo untreated control under drought stress, reaching 0.19 and 0.20 mm, respectively. These results indicate that α-Lipoic acid concentration and irrigation status may have different effects on leaf thickness values depending on the rootstock type and 1103 P had higher mean leaf thickness values, especially under drought stress and 50 μM α-Lipo, while 3309 C under the same conditions provided higher leaf thickness values between 25–50 μM α-Lipo concentrations. The lowest leaf thickness averages were generally obtained from 0 and 50 μM α-Lipo concentrations in well-irrigated conditions and 0 μM α-Lipo treatment in drought conditions for both rootstocks (0.15–0.17 mm). However, the difference between leaf thickness values of 1103 P and 3309 C at all α-Lipo concentrations under both irrigated and drought conditions (except for the 50 μM α-Lipo treatment under drought conditions) was not significant. The highest averages in terms of leaf area were obtained from 1 and 25 μM α-Lipo treatments of 1103 P under well-irrigated conditions (67.96 and 63.26 cm², respectively) and from 25 and 50 μM α-Lipo treatments of 3309 C under the same conditions (64.08 and 64.73 cm², respectively) (Figure 3C, Table S5). 25 μM α-Lipo application was the common α-Lipo concentration resulting in high leaf area values in both rootstocks under well-irrigated conditions, while 1 μM α-Lipo application showed a more favourable effect in 1103 P and 50 μM α-Lipo application gave better results in 3309 C. These results indicate that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type, and that 1103 P showed the best performance, especially under well-irrigated conditions and low or medium concentrations of α-Lipo, while 3309 C reached the highest values with medium or high concentrations of α-Lipo under the same conditions. In both rootstocks under drought stress, 0 and 1 μM α-Lipo treatments showed the lowest leaf area values (24.05–29.33 cm²). Except for 1 and 50 μM α-Lipo treatments under well-irrigated conditions, the difference between the leaf area values of 1103 P and 3309 C was found insignificant under all other irrigation conditions and α-Lipo concentrations.

3.2 The effect of α-lipoic acid application on some physiological, and biochemical parameters in rootstocks under drought stress

The highest averages for chlorophyll content were obtained from 1 and 50 μM α-Lipo treatments of 1103 P under well-irrigated conditions (45.41 and 43.81 SPAD, respectively) and 25 and 50 μM α-Lipo treatments of 3309 C under the same conditions (45.52 and 45.78 SPAD, respectively) (Figure 3D, Table S6). Here, 50 μM α-Lipo application was the common α-Lipo concentration, resulting in high chlorophyll content values in both rootstocks under well-irrigated conditions, while 1 μM α-Lipo application showed a more favourable effect in 1103 P and 25 μM α-Lipo application gave better results in 3309 C. These results suggest that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type. Interestingly, in contrast to the previous parameters, the 25 μM α-Lipo treatment did not show the highest effect at 1103 P under well-irrigated conditions, but this treatment showed higher averages than the control without α-Lipo treatment and was in the same statistical group with the 50 μM α-Lipo treatment. In terms of chlorophyll content, the lowest mean value of 25.27 SPAD was obtained from 0 μM α-Lipo treatment of 3309 C under drought conditions, followed by 1103 P under the same conditions with a SPAD index of 29.03. The difference between the SPAD index values of 1103 P and 3309 C in all other irrigation conditions and α-Lipo concentrations was found to be insignificant except for 1 and 25 μM α-Lipo applications in well-irrigated conditions and 0 μM α-Lipo application in drought conditions. In 1103 P, the RWC value was highest (90.46–91.54%) at all α-Lipo concentrations in well-irrigated conditions (Figure 3E, Table S6). At 3309 C, a different trend was observed, with the highest effects of 90.08 and 88.14% obtained from 25 and 50 μM α-Lipo treatments in well-irrigated conditions, respectively, whereas the effect of 1 μM α-Lipo in the same conditions was lower (87.71%). These results indicate that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type, and 1103 P showed high performance at all α-Lipo concentrations under well-irrigated conditions, while 3309 C provided the highest values under well-irrigated conditions and α-Lipo applied in the range of 25–50 μM. Both rootstocks under drought stress had lower RWC averages when α-Lipo was not applied. 3309 C rootstock under drought stress and without α-Lipo treatment exhibited the lowest values in terms of RWC with 55.28%. This value was followed by 1103 P rootstock under the same conditions with 65.08%. Under both well-irrigated conditions and drought stress, 1103 P had higher RWC compared to 3309 C when α-Lipo was not applied. A similar situation was observed at 1 μM α-Lipo concentration under well-irrigated conditions.

In terms of stomatal conductance value, the highest averages of 113.01 mmol m⁻² s⁻¹ were obtained from 1 μM α-Lipo concentration of 1103 P under well-irrigated conditions, while the highest effects of 107.30 mmol m⁻² s⁻¹ were obtained from 25 μM α-Lipo application under well-irrigated conditions at 3309 C, whereas the effect of 1 μM α-Lipo under the same conditions was much lower at 3309 C (87.71 mmol m⁻² s⁻¹) (Figure 3F, Table S6). These results suggest that α-Lipoic acid concentration and irrigation status may have different effects depending on the rootstock type and that 1103 P showed higher performance at lower levels of α-Lipo concentration under well-irrigated conditions, while 3309 C provided higher stomatal conductance values at moderate α-Lipo concentration under the same conditions. Both rootstocks under drought stress had lower stomatal conductance values when α-Lipo was not applied. 3309 C rootstock under drought stress and without α-Lipo treatment exhibited the lowest stomatal conductance value of 38.40 mmol m⁻² s⁻¹. This value was in the same statistical group with 1103 P rootstock under the same conditions with 38.50 mmol m⁻² s⁻¹ and 3309 C under drought conditions with 1 μM α-Lipo concentration. Under well-irrigated conditions, 1103 P had higher stomatal conductance than 3309 C at 0, 1, and 50 μM α-Lipo concentrations. Similar situation was observed in 50 μM α-Lipo treatment under drought conditions. The lowest leaf temperature value of 23.77°C was obtained from 25 μM α-Lipo treatment of 3309 C, while the highest averages were obtained from 0 μM α-Lipo concentrations (1103 P: 25.70°C and 3309 C: 25.87°C) and 1 μM α-Lipo treatment of 3309 C (25.63°C) under drought conditions in both rootstocks (Figure 4A, Table S7). Interestingly, the 50 μM α-Lipo treatment of 3309 C under well-irrigated conditions reached 25.24°C, a significant deviation from the other concentrations of 3309 C under the same conditions. However, 25 and 50 μM α-Lipo concentrations under well-irrigated conditions and 1 μM α-Lipo treatment under drought conditions were the treatments in which leaf temperature values of both rootstocks differed from each other. In 1103 P, the electrolyte leakage values of α-Lipo treatments at 0 and 1 μM concentrations under well-irrigated conditions were significantly lower and provided the lowest averages of 12.39 and 13.25%, respectively (Figure 4B, Table S7). At 3309 C, a similar trend was observed, and the lowest electrolyte leakage values were obtained from 0 and 1 μM α-Lipo treatments under well-irrigated conditions with 13.31 and 13.96%, respectively. In both rootstocks under drought stress, higher electrolyte leakage values were observed when α-Lipo was not applied. 3309 C rootstock under drought stress and without α-Lipo treatment showed the highest electrolyte leakage values with 19.63%. This value was followed by 1103 P rootstock under the same conditions with 17.56%. Under both well-irrigated conditions and drought stress, 1103 P showed lower electrolyte leakage compared to 3309 C when α-Lipo was not applied. A similar situation occurred at 25 μM α-Lipo concentration under drought conditions. The lowest MDA levels were obtained from all α-Lipo concentrations (17.80–18.67 nmol g⁻¹) of 1103 P under well-irrigated conditions (Figure 4C, Table S7). A different trend was observed in 3309 C, where the lowest MDA levels of 27.81 nmol g⁻¹ were obtained from 0 μM α-Lipo, while α-Lipo concentrations in the range of 1–50 μM led to an increase in MDA values (30.74–31.16 nmol g⁻¹). Both rootstocks under drought stress had higher MDA averages when α-Lipo was not applied. 3309 C rootstock under drought stress and without α-Lipo treatment exhibited the highest values in terms of MDA with 165.09 nmol g⁻¹. This value was followed by 1103 P rootstock under the same conditions with 150.14 nmol g⁻¹. 1103 P showed lower MDA levels than 3309 C at all α-Lipo concentrations under both well-irrigated conditions and drought stress.

The lowest SOD activity was obtained from 0 μM α-Lipo concentration (83.13 U mg⁻¹ protein) of 3309 C under well-irrigated conditions (Figure 4D, Table S8). This was followed by 1 and 25 μM α-Lipo treatments of 3309 C in the same conditions (88.33 and 89.17 U mg⁻¹ protein, respectively). In 1103 P, the lowest SOD activity was 101.81 U mg⁻¹ protein from 0 μM α-Lipo, followed by 1 μM α-Lipo concentration (104.13 U mg⁻¹ protein) of the same rootstock under the same conditions. Both rootstocks under drought stress showed lower SOD activity when α-Lipo was not applied. Drought-stressed rootstock 1103 P treated with 50 μM α-Lipo exhibited the highest levels of SOD activity with 156.30 U mg⁻¹ protein. This value was followed by 3309 C rootstock under the same conditions with 140.10 U mg⁻¹ protein. Under both well-irrigated conditions and drought stress, 1103 P showed higher SOD activity than 3309 C at all α-Lipo concentrations (except for 0 μM α-Lipo treatment under drought conditions). The lowest values in terms of CAT activity were obtained as 0.06 U mg⁻¹ protein from 0 μM α-Lipo application of 3309 C under well-irrigated conditions, followed by 1103 P application (0.10 U mg⁻¹) under the same conditions (Figure 4E, Table S8). Both rootstocks under drought stress showed lower CAT activity when α-Lipo was not applied. In contrast, 50 μM α-Lipo treatment showed the highest CAT activity with 0.95 U mg⁻¹ protein in 1103 P rootstock under drought stress. This value was followed by 3309 C rootstock under the same conditions with 0.74 U mg⁻¹ protein. Under drought stress conditions, 1103 P rootstock had higher CAT activity compared to 3309 C at all α-Lipo concentrations. On the other hand, the difference between rootstocks 1103 P and 3309 C was insignificant at 1–50 μM α-Lipo treatments under well-irrigated conditions. The lowest APX activity was obtained from 0 μM α-Lipo concentrations (0.37 and 0.16 U mg⁻¹ protein, respectively) of 1103 P and 3309 C under well-irrigated conditions (Figure 4F, Table S8). APX activity exhibited highly parallel responses to different α-Lipo concentrations in both rootstock 1103 P and 3309 C under well-irrigated conditions. Both rootstocks under drought stress showed lower APX activity when α-Lipo was not applied. The 1103 P rootstock under drought stress exhibited the highest APX activity with 9.81 U mg⁻¹ protein when treated with 50 μM α-Lipo. This value was followed by 3309 C rootstock with 8.93 U mg⁻¹ protein under the same conditions. Under drought stress, 1103 P showed higher APX activity than 3309 C at all α-Lipo concentrations.

The lowest proline content was obtained from all α-Lipo concentrations (0.27–0.29 μmol g⁻¹) of 1103 P and 3309 C under well-irrigated conditions (Figure 5A, Table S9). Proline accumulation showed very similar levels against different α-Lipo concentrations in both rootstock 1103 P and 3309 C under well-irrigated conditions. Both rootstocks under drought stress showed lower proline levels when α-Lipo was not applied. The 1103 P rootstock under drought stress showed the highest proline accumulation with 0.84 μmol g⁻¹ when treated with 50 μM α-Lipo. This value was followed by 3309 C rootstock with 0.79 μmol g⁻¹ under the same conditions. Under drought stress, 1103 P exhibited higher proline accumulation than 3309 C at all α-Lipo concentrations. The lowest protein content was obtained from 0 μM α-Lipo concentrations of 1103 P and 3309 C under well-irrigated conditions (1.31 and 1.30 mg, respectively) and 0 μM α-Lipo treatment of 3309 C under drought stress (1.40 mg) (Figure 5B, Table S9). In contrast, the highest values were obtained from 1 μM α-Lipo concentration of 1103 P under well-irrigated conditions (2.02 mg) and 50 μM α-Lipo treatment of the same rootstock under drought stress conditions (1.95 mg). These values were in the same statistical group with 2.00 mg obtained from 25 μM α-Lipo concentration of 3309 C under well-irrigated conditions. In 1103 P under well-irrigated conditions, the effect of low α-Lipo concentrations on protein accumulation was higher, while the effect of medium and high concentrations was lower compared to 1 μM α-Lipo. On the other hand, in 3309 C under the same conditions, the highest effects were obtained from 25 μM α-Lipo, while the effects of 1 and 50 μM α-Lipo applications on protein accumulation were lower. This indicates that the rootstocks used may vary significantly depending on the irrigation status and α-Lipo concentration. On the other hand, a similar trend was observed in both rootstocks under drought stress conditions and a significant increase in protein content occurred in parallel with increasing α-Lipo concentrations. This increase in protein content under drought conditions was more pronounced in 1103 P treated with 0 and 50 μM α-Lipo, while the levels were quite similar between 1103 P and 3309 C at 1 and 25 μM α-Lipo concentrations.

3.3 General evaluation of morphological physiological and biochemical responses

Principal Component Analysis (PCA) was performed to visualize the direction and strength of the relationship between different α-Lipo concentrations, irrigation status and rootstocks (Figure 6). The first two components explained most of the total data variance (about 90%). This shows that the PCA analysis of the components largely preserves the information in the original data set and summarises the structure of the data set in a comprehensible way. Biplot effectively discriminated between well-irrigated, and drought stressed plants and utilized the Principal Component 2 (PC2) axis for this purpose. Biplot revealed a strong negative correlation between plant growth traits (shoot and root length, shoot fresh and dry weight, root fresh and dry weight, number of leaves and leaf area) and oxidative stress parameters (leaf temperature, electrolyte leakage and malondialdehyde). In contrast, plant growth traits showed a strong positive correlation among themselves. A similar strong and positive correlation was found between antioxidant enzymes (SOD, CAT and APX) and proline. On the other hand, there was a strong positive correlation between leaf temperature, electrolyte leakage and leaf temperature. PC1 is explaining 68. The best-represented treatments in terms of oxidative stress parameters on the positive axis of PC1 (Principal Component 1), which explained 68.90% of the total variance, were 1103 P (R1-DS-0) and 3309 (R2-DS-0) without α-Lipo treatment under drought stress conditions, while the treatments that were closest to these treatments and therefore had the lowest potential to alleviate drought stress were 1 μM α-Lipo treatments of 3309 C and 1103 P under drought conditions (R2-DS-1 and R1-DS-1, respectively). The best-represented treatments in terms of plant growth traits and photosynthetic and osmotic parameters (chlorophyll content, stomatal conductance and RWC) on the negative axis of the same component were 1 μM α-Lipo treatment of 3309 C under well-irrigated conditions (R2-WI-1), respectively, 25 μM α-Lipo application of 3309 C under the same conditions (R2-WI-25) and 25 μM α-Lipo application of 1103 P under well-irrigated conditions (R1-WI-25) and 50 μM α-Lipo application of 1103 P under the same conditions (R1-WI-50). PC2 explaining 20. The best-represented treatments in terms of protein content, leaf thickness, proline content and antioxidant enzymes, which have a larger load value on the positive axis of PC2, explaining 20.90% of the total variance, were 50 μM α-Lipo treatments of 1103 P and 3309 C under drought conditions (R1-DS-50 and R2-DS-50, respectively), 25 μM α-Lipo treatment of 1103 P under the same conditions (R1-DS-25) and 25 μM α-Lipo treatment of 3309 C under the same conditions (R2-DS-25).

Heatmap analysis to visualize the data shows the responses of grapevine rootstocks to irrigation status and different α-Lipo concentrations and how these responses vary between different groups (Figure 7). Heatmap analysis significantly overlapped with Biplot in terms of the separation of treatments. Heatmap basically categorized the treatments into two main clusters. The first main cluster included drought-stressed practices, while the second main cluster included well-irrigated practices. In the first main cluster, drought stress treatments were divided into two sub-clusters. In the first subset, 50 μM α-Lipo treatment (R1-DS-50) of 1103 P under drought conditions, which showed the highest levels in terms of plant growth characteristics, photosynthetic and osmotic parameters but the lowest levels in terms of oxidative stress parameters and thus had the highest tolerance to drought stress, was included and showed a separate branching and separated from the other treatments and formed the first group. In the second group belonging to the same subset, 50 μM α-Lipo treatment of 3309 C under drought conditions (R2-DS-50) and 25 μM α-Lipo treatment of 1103 P under the same conditions (R1-DS-25) were included. These treatments also had a strong potential for drought tolerance. The second subset was divided into two subgroups, the first subgroup included 1 μM α-Lipo treatments of 1103 P and 3309 C under drought conditions (R1-DS-1 and R2-DS-1, respectively), while 25 μM α-Lipo treatment of 3309 C under the same conditions (R2-DS-25) was also included in this group. The second group of the second subset included 0 μM α-Lipo treatments of 1103 P and 3309 C under drought conditions (R1-DS-0 and R2-DS-0, respectively). The second main cluster essentially divided the treatments into two sub-clusters. The first subset included treatments with lower means for plant growth characteristics photosynthetic and osmotic parameters. Here, the 0 μM α-Lipo treatment (R1-WI-0) of 1103 P under well-irrigated conditions showed a distinct branching with its relatively higher potential and formed the first group. In the second group, 0 and 1 μM α-Lipo treatments of 3309 C under the same conditions (R2-WI-0 and R2-WI-1, respectively) were included. In the second subset, 50 μM α-Lipo treatment (R2-WI-50) of 3309 C under well-irrigated conditions formed the first group. The 1, 25 and 50 μM α-Lipo treatments of 1103 P (R1-WI-1, R1-WI-25 and R1-WI-50, respectively) and 25 μM α-Lipo treatment of 3309 C (R2-WI-25) under well-irrigated conditions, which showed the highest levels in terms of plant growth characteristics, photosynthetic and osmotic parameters, but the lowest levels in terms of oxidative stress parameters, formed the second group.

4.1 The effect of α-lipoic acid application on some morphological parameters in rootstocks under drought stress

The findings of our comprehensive analysis underscore the significant superiority of the 1103 P rootstock over 3309 C across a broad spectrum of growth and physiological parameters (Figure 2-5, Table S3-9), notably under the influence of α-lipoic acid (α-Lipo) treatments. The exogenous application of α-Lipo exhibited significant effects on various growth parameters in the grapevine rootstocks ‘1103 P' and ‘3309 C' under well-irrigated and drought conditions. The responses were dose-dependent and influenced by the rootstock type and irrigation status, indicating a complex interplay between these factors, as previously observed in other plant species (Navari-Izzo et al., 2002; Guler et al., 2021; Gorcek & Erdal, 2015; Shay et al., 2009; Javeed et al., 2021). Under well-irrigated conditions, the ‘1103 P' rootstock exhibited superior performance in terms of shoot length, shoot fresh and dry weight, root length, root fresh and dry weight, leaf number, and leaf area when treated with lower concentrations of α-Lipo, particularly 1 μM. This suggests that ‘1103 P' has a higher sensitivity and responsiveness to lower levels of α-Lipo, which could be attributed to its inherent physiological and genetic characteristics (Lanoue et al., 2017). Conversely, the ‘3309 C' rootstock demonstrated optimal responses in these parameters at higher α-Lipo concentrations, ranging from 25 to 50 μM. Under drought stress conditions, both rootstocks exhibited lower values for the measured parameters compared to well-irrigated conditions, indicating the detrimental effects of water deficit on plant growth and development. However, the application of α-Lipo mitigated these adverse effects to some extent, with higher concentrations (25–50 μM) being more effective in alleviating drought stress, particularly in the ‘3309 C' rootstock. The observed differences in the responses of the two rootstocks to α-Lipo treatments under varying irrigation conditions suggest that the efficacy of α-Lipo is modulated by the genetic makeup and inherent stress tolerance mechanisms of the rootstocks, as previously documented (Akram et al., 2017; Nawaz et al., 2015; Lenk et al., 2019). Notably, the parameter of leaf thickness exhibited a unique response pattern, with the ‘1103 P' rootstock displaying higher values under drought stress and 50 μM α-Lipo treatment, while the ‘3309 C' rootstock showed higher leaf thickness at intermediate α-Lipo concentrations (25–50 μM) under the same conditions. This divergent response could be attributed to the differential regulation of leaf morphology and anatomical adaptations in these rootstocks as part of their drought stress response strategies. The variance observed in the responses of the two rootstocks indicates the pivotal role of rootstock selection in enhancing grapevine tolerance to drought, a critical concern in viticulture, given the escalating challenges of climate change. The differential responses to α-Lipo treatments, particularly the pronounced benefits observed with the non-stressed and α-Lipo-nontreated condition and escalated with increased α-Lipo concentrations, resonate with the findings of Navari-Izzo et al. (2002) and Guler et al. (2021), highlighting α-Lipo's potent antioxidant capabilities that mitigate oxidative stress and support plant growth under adverse conditions. Drawing from the observed dose-dependent improvement in parameters such as shoot length and leaf area with increasing α-Lipo concentrations, we hypothesize that α-Lipo enhances physiological tolerance and growth through its dual-phase antioxidant action, as suggested by Navari-Izzo et al. (2002). This assumption is further corroborated by similar findings of Gorcek and Erdal (2015), where α-Lipo treatments ameliorated the adverse effects of water and salt stress, respectively, by improving water status and leaf surface area, thereby facilitating better gas exchange and photosynthesis. In addition, the unique response of the ‘1103 P' and ‘3309 C' rootstocks to α-Lipo, despite their inherent differences in drought tolerance, indicates the adaptive mechanisms induced by α-Lipo treatments observed in other crops subjected to stress conditions, as discussed by Shay et al. (2009) and Javeed et al. (2021). These studies confirm the notion that exogenous application of growth regulators like α-Lipo can activate similar stress mitigation pathways in plants, irrespective of their innate stress tolerance levels. In light of our findings, we posit that the application of α-Lipo may serve as a strategic intervention to bolster grapevine tolerance against drought stress, enhancing both growth and physiological health. This hypothesis aims to bridge the gap in understanding how α-Lipo treatments modulate plant responses to abiotic stressors, offering insights into the underlying mechanisms of stress tolerance enhancement. By elucidating these relationships, our study contributes to the development of more sustainable and resilient viticultural practices, spotlighting the potential of α-Lipo as a valuable tool in the adaptive management of grapevines under the growing threat of climate-induced drought conditions.

4.2 The effect of α-lipoic acid application on some physiological, and biochemical parameters in rootstocks under drought stress

The study investigated the effects of various concentrations of α-lipoic acid (α-Lipo) on physiological and biochemical parameters of two different rootstocks (1103 P and 3309 C) under well-irrigated and drought stress conditions. Our study elucidated the multifaceted role of α-Lipo in augmenting drought stress tolerance, with implications for improving agricultural tolerance to climate variability. The 1103 P rootstock exhibited superior physiological and biochemical responses to drought stress and α-Lipo treatment compared to the 3309 C rootstock. Specifically, 1103 P demonstrated higher chlorophyll content (Figure 3D), stomatal conductance (Figure 3F), relative water content (RWC) (Figure 3E), and antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT; and ascorbate peroxidase, APX) (Figures 4D-F), alongside lower electrolyte leakage (Figure 4B), leaf temperature (Figure 4A), and malondialdehyde (MDA) levels (Figure 4C). These findings are aligned with the hypothesis that certain rootstocks possess inherent qualities that enhance their drought tolerance and response to antioxidative treatments (Mihailović et al., 1997; Elkelish et al., 2021). The higher chlorophyll content observed in 1103 P suggests its better capacity to maintain chlorophyll integrity under stress, consistent with improved photosynthetic pigment biosynthesis and reduced chlorophyll degradation (Mihailović et al., 1997; Elkelish et al., 2021). The capacity of α-Lipo to enhance chlorophyll content indicates its role in mitigating oxidative damage and supporting photosynthetic activity. The increased stomatal conductance and RWC in α-Lipo-treated plants, particularly in 1103 P, are indicators of improved water status and ability to regulate water loss under drought conditions (Cornic, 2000; Flower & Ludlow, 1986). The positive effect of α-Lipo on these parameters may be attributed to its influence on abscisic acid signalling, leading to optimized stomatal opening and improved water use efficiency. The lower electrolyte leakage, leaf temperature, and MDA levels in 1103 P suggest a more robust membrane stability and reduced lipid peroxidation, indicative of effective antioxidative defence mechanisms (Elkelish et al., 2021; Terzi et al., 2018). The significant reduction in MDA content with α-Lipo treatment further supports its role in enhancing plant tolerance to oxidative stress. The notably higher antioxidant enzyme activities (SOD, CAT, APX) in 1103 P point to a more dynamic antioxidative response to neutralize reactive oxygen species (ROS) (Ibrahim et al., 2021; Ramadan et al., 2022). The increased enzyme activities upon α-Lipo treatment show α-Lipo's potential to modulate antioxidant defence systems, consistent with findings from Gorcek and Erdal (2015). The differential responses between 1103 P and 3309 C rootstocks, especially under α-Lipo treatment, shed light on the genetic and physiological nuances influencing plant adaptation to drought stress. The findings suggest that 1103 P's inherent traits, combined with α-Lipo's antioxidative properties, confer enhanced drought tolerance through improved water status, reduced oxidative damage, and elevated antioxidative enzyme activities. Especially, the 50 μM dose of α-Lipo was identified as the most effective in enhancing physiological and biochemical responses in both 1103 P and 3309 C rootstocks under drought stress. This dosage improved chlorophyll content, stomatal conductance, RWC, and antioxidant enzyme activities (SOD, CAT, APX), while significantly reducing electrolyte leakage, leaf temperature, and MDA levels. These findings align with previous research suggesting that higher doses of α-Lipo can support photosynthetic pigment biosynthesis, reduce oxidative damage, and positively affect plant growth and development (Terzi et al., 2018; Elkelish et al., 2021; Sezgin et al., 2019). Furthermore, the effectiveness of the 50 μM α-Lipo dose can be associated with its modulatory effect on antioxidant enzymes critical for protecting plants against oxidative damage under drought stress (Huang et al., 2019; El-Beltagi et al., 2022). Research has demonstrated that α-Lipo can increase the activities of key antioxidant enzymes such as SOD, CAT, and APX, providing an effective defence against ROS.

4.3 General evaluation of morphological physiological and biochemical responses

The principal component analysis (PCA) and heatmap interpretations of our study offer profound insights into the morphological, physiological, and biochemical responses of grapevine saplings to drought stress and α-lipoic acid (α-Lipo) treatments. The significant capture of overall variance by the first two principal components in the PCA (Figure 6) shows the complex interplay between oxidative stress markers and growth-related parameters, as well as the antioxidative defence mechanisms within the plants. The strong negative correlation of the first principal component with growth parameters and photosynthetic attributes against oxidative stress markers such as electrolyte leakage, MDA content, and leaf temperature delineates the detrimental impact of drought stress on plant vitality. These findings are consistent with extensive research showing that drought stress precipitates an increase in oxidative stress markers, leading to cellular damage and a consequent reduction in growth and photosynthetic efficiency (Navari-Izzo et al., 2002). The adverse effects of drought stress, characterized by the elevation of electrolyte leakage, MDA, and leaf temperature, reflect the disruption of membrane integrity and the accumulation of oxidative damage within cellular structures, which are commonly observed under such stress conditions (Elkelish et al., 2021; Terzi et al., 2018). Conversely, the positive correlation of the second principal component with proline levels and activities of antioxidative enzymes (, SOD, CAT and APX) alongside leaf thickness suggests a robust antioxidative defence mechanism in response to drought stress. The accumulation of proline and the upregulation of antioxidative enzymes are well-documented adaptive responses to drought, acting to mitigate oxidative damage and stabilize cellular structures (Shay et al., 2009; Javeed et al., 2021). Proline, in particular, functions as an osmoprotectant and a scavenger of free radicals, while SOD, CAT, and APX play critical roles in the detoxification of ROS, thereby preserving cellular integrity under stress conditions. The proximity of 50 μM α-Lipo treatments to the positive control samples in the PCA biplot, as well as their grouping with positive controls in the heatmap analysis (Figure 7), signifies the efficacy of higher α-Lipo concentrations in ameliorating drought-induced stress. This observation suggests that α-Lipo, particularly at higher doses, can simulate conditions akin to optimal growth environments by enhancing the antioxidative defence systems and mitigating the physiological and biochemical impacts of drought stress. This is consistent with the findings of Gorcek and Erdal (2015), who reported the ameliorative effects of α-Lipo on water and salt stress through improvements in water status, antioxidative enzyme activities, and overall plant growth. Moreover, the distinct grouping of treatments based on α-Lipo concentration in the heatmap indicates the dose-dependent response of grapevine saplings to α-Lipo, with 50 μM treatments markedly enhancing physiological states in contrast to the minimal effects observed with 1 μM treatments. This dose–response relationship highlights the potential of α-Lipo as a critical agent in modulating plant responses to abiotic stressors, offering a strategic intervention to enhance grapevine tolerance against drought stress.