Overexpression of Potato PYL16 Gene in Tobacco Enhances the Transgenic Plant Tolerance to Drought Stress

Abstract

PYR/PYL/RCAR proteins are abscisic acid (ABA) receptors that play a crucial role in plant responses to abiotic stresses. However, there have been no research reports on potato PYL so far. In this study, a potato PYL gene named StPYL16 was identified based on transcriptome data under drought stress. Molecular characteristics analysis revealed that the StPYL16 protein possesses an extremely conserved PYL family domain. The tissue expression results indicated that the StPYL16 is predominantly expressed at high levels in the underground parts, particularly in tubers. Abiotic stress response showed that StPYL16 has a significant response to drought treatment. Further research on the promoter showed that drought stress could enhance the activation activity of the StPYL16 promoter on the reporter gene. Then, transient and stable expression of StPYL16 in tobacco enhanced the drought resistance of transgenic plants, resulting in improved plant height, stem thickness, and root development. In addition, compared with wild-type plants, StPYL16 transgenic tobacco exhibited lower malondialdehyde (MDA) content, higher proline accumulation, and stronger superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. Meanwhile, StPYL16 also up-regulated the expression levels of stress-related genes (NtSOD, NtCAT, NtPOD, NtRD29A, NtLEA5, and NtP5CS) in transgenic plants under drought treatment. These findings indicated that the StPYL16 gene plays a positive regulatory role in potato responses to drought stress.

1. Introduction

Plant growth and development are frequently compromised by a variety of abiotic stresses, including cold, drought, and salinity. Abiotic stress is a major hidden danger in agricultural production, commonly resulting in reduced crop yields [1]. Following exposure to abiotic stress, plants need to orchestrate physiological and biochemical responses, along with gene regulation, to acclimate to adverse environmental conditions [2]. Gene regulation is intricately linked to stress-induced hormonal signaling pathways. Studies have demonstrated that the plant hormone ABA plays a pivotal role in mediating plants’ responses to abiotic stress [3].

The ABA signal is initially recognized and triggered by the presence of ABA, with ABA perception achieved through the binding of ABA receptors to the hormone [4]. The predominant type of ABA receptors is the pyrabactin resistance (PYR)/PYR-like (PYL)/regulatory component of the ABA receptor (RCAR) complex, which localizes to the nucleus and cytosol [4]. The PYR/PYL/RCAR (referred to hereafter as PYL) ABA receptor, in conjunction with protein phosphatase 2C (PP2C) and SNF1-related protein kinase 2 (SnRK2), forms the core ABA signaling network, which features a dual negative regulatory mechanism [4,5]. In the absence of ABA, PP2Cs bind to SnRK2s, thereby suppressing the activity of SnRK2 proteins. Upon an elevation in ABA levels, ABA is recognized and bound by its receptors, initiating interaction between the ABA receptor complex and PP2Cs. Consequently, SnRK2s are released from inhibition, leading to the activation of downstream target gene expression [5,6]. In plants, numerous ABA receptors, protein phosphatases, and kinases have been recognized as pivotal elements in the ABA signaling pathway. For instance, Arabidopsis possesses 14 PYR/PYL receptors, 76 PP2C phosphatases, and 10 SnRK2 kinases [7,8]. Following research on Arabidopsis, members of the PYL, PP2C, and SnRK2 families have also been identified in other plants such as rice [9], maize [10,11], and tomato [12].

As a stress hormone, the concentration of ABA in plants increases rapidly under abiotic stress conditions, particularly drought and salt stress [5,13]. In recent years, the role of ABA receptors in plant responses to abiotic stress has been gradually elucidated. Overexpression of AtPYL4/RCAR10, AtPYL5/RCAR8, and AtPYL13/RCAR10 enhanced the drought resistance of transgenic Arabidopsis [14,15]. Meanwhile, AtPYL9/RCAR1 enhanced drought resistance through the promotion of leaf senescence, thereby reducing transpiration and facilitating water transport to young tissues [15]. Furthermore, ABA receptors AtPYL1/RCAR12 and AtPYL3/RCAR13 in Arabidopsis have been identified to play crucial roles in extreme temperature responses, with their overexpression in plants demonstrating significant tolerance to cold and heat stress [16]. The functional role of PYL genes has been observed in a variety of food and commercial crops. For example, overexpression of certain PYL genes, such as OsPYL3, 9, and 10, enhances drought and cold tolerance in transgenic rice [17,18]. Similarly, overexpression of OsPYL5 improves drought and salt tolerance in rice [19]. In wheat, overexpression of TaPYL4 enhances grain production under drought conditions and increases water-use efficiency [20]. PYL family genes have also been identified in fruit crops like grape [21], sweet orange [22], banana [23], and apple [24]. However, research on the biological functions and regulatory roles of these genes in the stress responses of potatoes is limited.

The potato (Solanum tuberosum L.) plays a vital role in global food security as the fourth largest crop worldwide. However, it often encounters various stresses, especially drought, during growth in regions like Gansu, China, known for low precipitation [25]. Our team focuses on developing drought-resistant potato varieties, identifying drought-resistant genes, and analyzing regulatory mechanisms. Progress has been achieved in studying the hormone regulation of potato growth and stress responses. However, the characterization of potato PYL genes and their functions in drought stress responses remain largely unexplored. Based on transcriptome data from drought-stressed potatoes, we identified a significant PYL gene responsive to drought stress and designated it as StPYL. Through scrutinizing the reaction of the StPYL16 gene and its promoter to drought stress, the drought resistance capacity of StPYL16 was subsequently validated through overexpression in tobacco. This pioneering research delves into the role of the PYL genes within potatoes, showcasing pivotal implications in unraveling the ABA signaling pathway in potatoes amidst drought challenges.

2. Results

2.1. Screening and Molecular Characterization Analysis of StPYL16

PYL serves as a crucial ABA receptor in plant resistance against abiotic stresses, such as drought. Consequently, our team conducted a thorough analysis of the potato PYL gene family at an early stage, identifying a total of 23 potato PYL gene sequences (Figure S1). Subsequently, we examined the expression profiles of all PYL genes in two potato varieties with varying levels of drought resistance using transcriptome data. This analysis aimed to pinpoint the key PYL genes that play a role in the potato’s response to drought stress. Ultimately, we identified the gene PGSC0003DMT400003773, which exhibited a significant response to drought stress, and designated it as StPYL16 (Figure S2). Tissue-specific expression analysis revealed that StPYL16 is predominantly expressed in the below-ground organs, encompassing the young tuber, mature tuber, tuber pith, tuber peel, tuber sprout, and tuber cortex. Conversely, in the above-ground organs, its expression is notably elevated in the stem while relatively subdued in other tissues (Figure 1).

2.2. StPYL16 Is Strongly Induced by Drought Treatment

In order to clarify the response of StPYL16 to abiotic stress, its expression levels under ABA and drought treatment were further analyzed by qRT-PCR (Figure 2). Two varieties, Atl (drought sensitive) and Q9 (drought tolerant), were chosen for the study. Under ABA treatment conditions, StPYL16 exhibited a pronounced response in QS. Significant differences were observed after 1 h of stress, and its expression level gradually increased as the treatment duration extended, peaking at 12 h. At this point, the expression level was 7.98 times higher than that at 0 h. In contrast, In drought-sensitive materials, StPYL16 exhibited an inverse pattern, showing a significant decrease after 6 h of stress followed by stabilization. Under drought stress, StPYL16 demonstrated a similar expression pattern to that under ABA treatment in QS9, with a notable increase in expression from the onset of stress, reaching its peak after 12 h. In the Atl, there were significant differences in StPYL16 expression levels at 3 and 12 h of stress, but the overall expression level did not show a regular trend.

2.3. Drought Enhances the Activity of StPYL16 Promoter

In order to further elucidate the mechanism by which StPYL16 responds to stressful environments, a 2000 bp promoter fragment upstream of the StPYL16 gene start codon was obtained using TBtools. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 25 March 2024) was employed for predicting the regulatory elements within the promoter region. The findings showed that the identified cis-elements fall into two main categories according to their responsive functions: hormone-responsive elements and stress-responsive elements (Figure 3a). Furthermore, the promoter sequence also contains various other types of regulatory elements, such as photoresponsive elements and developmental-related elements.

To examine the response of the StPYL16 promoter to ABA and drought stress, the proStPYL16 sequence was isolated from potato genomic DNA and inserted into the pBI101–GUS vector for further analysis. Under normal conditions, transgenic plants containing proStPYL16 displayed a blue coloration in histological GUS assays, while the negative control exhibited a faint yellow color (Figure 3b). This observation suggested that proStPYL16 activates the downstream GUS reporter gene expression, indicating its intrinsic promoter strength. Subsequent exposure to drought stress resulted in a more intense GUS staining pattern in transgenic tobacco plants, suggesting an enhancement in the promoter activity of proStPYL16 under drought conditions. Conversely, no significant difference in GUS staining was observed between ABA treatment and normal conditions, implying that the proStPYL16 promoter may not be responsive to ABA. These findings collectively suggested that StPYL16 may function as a drought-responsive gene.

2.4. Transient Transformation of StPYL16 Improves Drought Tolerance of Transgenic Tobacco

To preliminarily investigate the function of StPYL16, we transiently expressed it in tobacco and subjected it to drought treatment to explore whether it can affect the drought resistance of transgenic tobacco. Initially, GUS staining results of the transgenic plants revealed that leaves transformed with empty vector and StPYL16 exhibited a distinct blue color, indicating successful introduction of the target gene (Figure 4a). Concurrently, overexpression of this gene resulted in a certain degree of improvement in the wilting degree of plants compared to the control, indicating that the expression of StPYL16 enhanced the drought resistance of tobacco (Figure 4b).

Moreover, to elucidate the impact of StPYL16 on transgenic plants, we assessed the levels of MDA and Pro, which are closely linked to plant stress resistance, as well as the activity of antioxidant enzymes (SOD, POD, and CAT). The findings demonstrated that over-expression of StPYL16 led to a substantial increase in Pro accumulation in transgenic plants under drought stress while decreasing the MDA content. Conversely, under normal conditions, no significant disparity was observed between the two indicators. The activity of antioxidant enzymes displayed a varied pattern. SOD and CAT activities were significantly higher under both control and stress conditions. POD activity exhibited a notable increase under drought stress, with no significant difference observed between transgenic and control plants under normal conditions (Figure 4c).

2.5. Generation of Stable Transgenic Tobacco

To further elucidate the function of StPYL16 under drought stress conditions, transgenic tobacco plants overexpressing StPYL16 were generated using the Agrobacterium-mediated leaf disk transformation method (Figure 5a). Fifteen transgenic plants with resistance were selected through kanamycin screening and qRT-PCR analysis (Figure 5b). The results revealed that StPYL16 was expressed in all resistant plants but not in the wild-type control plants. Consistent results were obtained from GUS histochemical staining (Figure 5c). Subsequently, we quantified the expression levels of StPYL16 in each positive plant using qRT-PCR and identified three transgenic lines (OE-4, OE-6, and OE-7) with high levels of StPYL16 expression for further investigation (Figure 5d).

2.6. Overexpression of StPYL16 Increases Tobacco Drought Tolerance

To investigate the phenotypic changes of transgenic tobacco before and after drought stress, photographs were taken of transgenic lines and wild-type plants under various treatments for observation. The findings indicated that under normal conditions, there was no notable distinction between each transgenic strain and the control plant. However, under stress conditions, the growth of all plants was impeded, with the control plants exhibiting a more remarkable inhibition. Specifically, at 100 mM, the plant height and root development of transgenic plants were significantly superior to those of the control. Conversely, at 200 mM and 300 mM, the root growth of all plants was completely suppressed (Figure 6a).

Further measurements were conducted on a variety of phenotypic indicators. The results revealed that the over-expression of StPYL16 did not impact the fresh weight of transgenic plants under drought conditions. Plant height, as a crucial indicator reflecting plant growth and development, was significantly higher in StPYL16 transgenic plants compared to control plants under 100 and 200 mM conditions. The height of the three transgenic lines nearly reached 1.5–1.8 times that of the control. Similarly, the number of leaves displayed a comparable pattern, with transgenic plants under 100 mM stress exhibiting significantly more leaves than the control, reaching 2.1 times the quantity (Figure 6b).

Furthermore, phenotype scans and related data indicator measurements were performed on the root systems of each plant (Figure 6c). The findings indicated that the root length of StPYL16 transgenic plants was notably greater than that of the control under drought stress. While other indicators such as root diameter, root surface area, and root volume showed some variances, these distinctions were not statistically significant (Figure 6d). Finally, two key physiological indicators closely associated with drought stress, MDA and Pro, were measured (Figure 7). Under normal conditions, no significant differences were observed between the lines. However, under drought conditions, the MDA content in each transgenic line was significantly lower than that in the control, whereas the Pro content was notably higher than that in the control. This disparity became more pronounced as the stress level intensified. In conclusion, the over-expression of StPYL16 significantly bolstered the drought resistance of transgenic tobacco.

2.7. Stress-Related Gene Expression in Transgenic Tobacco Plants under Drought Stress Mediated by StPYL16

To investigate the mechanism behind the enhanced drought tolerance in StPYL16 transgenic plants, we performed a comparative analysis of the expression patterns of stress-related genes in StPYL16 transgenic lines and control plants under both normal and drought conditions. Upon exposure to drought stress, certain stress-related genes, including NtSOD, NtCAT, NtPOD, NtRD29A, NtLEA5, and NtP5CS, exhibited significantly elevated expression levels in StPYL16 transgenic plants when compared to control plants (Figure 8). Nevertheless, there was no notable disparity in gene expression levels, except for the NtCAT gene, between StPYL16 transgenic plants and control plants that were grown under normal conditions. Notably, following drought stress, the expression levels of all examined genes in both wild-type and transgenic plants showed a marked increase compared to normal conditions (Figure 8). These findings suggested that the over-expression of StPYL16 may modulate the expression of these stress-related genes to bolster plant resilience against drought stress.

3. Discussion

The hormone ABA plays a crucial role in regulating plant growth, development, and stress responses [26,27]. Drought stress often leads to increased ABA synthesis, with the PYL protein playing a key role in mediating plant drought resistance through ABA signaling pathways [28]. Although the function of the PYL gene in improving plant drought tolerance has been thoroughly examined across a range of plant species, there is a lack of detailed functional studies on the specific role of PYL in potatoes.. This current research endeavor focuses on the cloning of StPYL16, a gene belonging to the PYL family, from potato plants. Subsequently, we conducted analyses to elucidate the regulatory role of StPYL16 in conferring drought resistance in transgenic tobacco plants following transient and stable expression.

With the continuous advancement of sequencing technology, an increasing number of plant genomes have been sequenced, facilitating the identification of gene families crucial for plant growth and stress response regulation within the entire genome. Despite the significant role of ABA in this process, it was not until 2009 that PYL was recognized as an ABA receptor through research involving the utilization of a synthetic ABA agonist known as pyranose [8]. Subsequent to this discovery, numerous studies have focused on the screening and identification of PYL family proteins across various plant species. In Arabidopsis thaliana, 14 PYL proteins were delineated into three subfamilies based on phylogenetic analysis as well as structural and functional characterization [29]. A genome-wide examination of plant PYL family proteins revealed variations in the number of PYL family genes among different plant species. For example, there are 15 in tomato [30], 14 in poplar [31], 40 in Gossypium hirsutum [32], 29 in tobacco [31], 8 in grape [21], 13 in rice [33], and 38 in wheat [34]. Despite the significant differences in the quantity of PLY family genes, the clustering pattern of these genes remains consistent across plant species. PYL family members in these plant species are classified into three subfamilies. In the current study, 23 PYL genes were identified in potato, a number similar to the 29 found in tobacco, possibly due to their classification within the Solanaceae family. Additionally, phylogenetic analysis demonstrated that potato PYL proteins were categorized into four subgroups, a slight deviation from previous reports suggesting most plant PYL proteins are divided into three subgroups. This difference may be associated with the complex chromosome structure of potatoes or the emergence of new genes with structural variances during evolution.

Drought, as a prominent abiotic stress factor, presents a formidable challenge to worldwide agricultural production, resulting in substantial annual decreases in crop yields. Plant hormones, notably ABA, play a pivotal role in plant responses to abiotic stress and the modulation of resistance mechanisms [26,27]. The PYL protein, serving as an ABA receptor, plays a vital part in ABA perception and signal transduction. Numerous studies have recognized its role in enhancing plant drought tolerance. For instance, the overexpression of AtPYR1, AtPYL1, AtPYL2, AtPYL3, AtPYL8, and AtPYL9 has been shown to significantly enhance the drought tolerance of transgenic Arabidopsis [15,35]. Similar results have been observed in other plant species, such as rice [36], corn [37], wheat [20], poplar [38], and cotton [39]. Through transcriptome analysis under drought stress in the early stages, we identified a member of the PYL gene, StPYL16, that may participate in the response to drought stress. Subsequent investigations involving promoter stress response detection and transgenic functional validation demonstrated that StPYL16 exerts a positive regulatory effect on potato response to drought stress (Figure 1). Similar to several PYL genes in previous studies, the overexpression of StPYL16 mitigates damage from drought stress, resulting in improved growth conditions in transgenic plants. Furthermore, the increased activity of antioxidant enzymes suggests that StPYL16 enhances plant drought tolerance by facilitating the removal of excess reactive oxygen species under stress conditions. Comparable outcomes have been reported in studies focusing on gene families in other plant species [40]. Further research is warranted to uncover the mechanisms through which PYL enhances antioxidant enzyme activity under stress conditions. Meanwhile, we examined the effect of ABA on StPYL. We found that the StPYL16 showed no significant response to exogenous ABA in two different drought-resistant varieties. Thus, our results suggested that StPYL16 are involved in plant stress resistance through an ABA-independent pathway.

When plants are subjected to drought stress, there is a disruption in the balance between intracellular activity production and clearance, leading to the accumulation of reactive oxygen species, which in turn causes oxidative damage and plant cell death. This phenomenon has been extensively studied and documented [41,42]. In the face of drought stress, plants elevate the transcription levels of antioxidant enzyme genes such as NtGPX, NtSOD, NtPOD, and NtCAT, leading to a subsequent escalation in the activity of SOD/CAT/POD enzymes [43,44]. These changes play a crucial role in enhancing plant drought resistance. In our study, it was found that overexpression of StPYL16 significantly boosted the expression of SOD/CAT/POD genes and the corresponding antioxidant enzyme activity in transgenic plants under drought stress conditions. Significantly, under standard growth conditions, the expression level of the CAT gene in StPYL16-overexpressing plants exhibited a marked increase compared to that in control plants. This observation was corroborated by the reduction in MDA content in transgenic plants (Figure 4), indicating reduced oxidative damage. The reactive oxygen species scavenging system is vital for plants to counteract drought stress, involving both enzymatic (such as superoxide dismutase, catalase, peroxidase, and GPX) and non-enzymatic scavengers. These enzymes work synergistically to mitigate the harmful effects of reactive oxygen species [44,45]. MDA, a byproduct of lipid oxidation, serves as a marker for plant oxidative damage [46]. Proline is an osmolyte found in plant cytoplasm that plays a crucial role in stabilizing membrane and protein conformation, clearing reactive oxygen species, and reducing photodamage to chloroplast thylakoid membranes [47]. In the current study, overexpression of StPYL16 led to an increase in free proline content in transgenic plants under drought stress conditions, corresponding to the upregulation of the proline synthesis gene NtP5CS (Figure 4). Proline biosynthesis involves the glutamate and ornithine pathways, utilizing glutamate and ornithine as precursors to catalyze the production of pyrroline-5-carboxylate (P5C) by P5CS and OAT enzymes, respectively [48]. The accumulation of proline in plants under abiotic stress conditions is typically linked to the upregulation of P5CS and OAT expression, along with the downregulation of proline dehydrogenase (ProDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) expression [49]. Hence, the findings suggested a positive correlation between the increased Pro levels in transgenic plants under drought stress and the enhanced expression of NtP5CS.

4. Materials and Methods

4.1. Plant Material and Growth Conditions

The potato varieties “Atlantic” and “Qingshu 9” along with tobacco “Nicotiana tabacum (NT12)” were stored at the State Key Laboratory of Aridland Crop Science, Gansu Agricultural University. Potato seedlings were cultivated in an artificial growth chamber with conditions of 16 h/8 h light/darkness, 25 °C, and 60% relative air humidity. Tobacco seeds were planted in a greenhouse with a temperature of 25 ± 2 °C, with a photoperiod of 12 h/12 h light/darkness and a relative humidity of 60%. The plant expression vectors pCAMBIA1304 and Agrobacterium tumefaciens GV3101 were kept in our laboratory.

4.2. Isolation and Characterization of StPYL16

Total RNA was extracted from the samples using an RNAout kit (Tiandz, Beijing, China) and then reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis kit (MBI, Woburn, MA, USA). Based on the transcriptome data of potato seedlings under drought stress, a novel PYL gene named StPYL16 was identified using specific primers. The gene sequence was analyzed using the NCBI database. Detailed information on the primers used can be found in Supplementary Table S1.

4.3. Expression Analysis of StPYL16

The tissue-specific expression of StPYL16 in potato and its reactions to various abiotic stresses were detected using qRT-PCR. Twenty days post-planting, potato seedlings were sprayed with 100 µM ABA or alcohol (as a control). For drought treatment, 20-day-old seedlings cultivated in untreated medium were shifted to liquid MS medium supplemented with 200 mM mannitol, with samples collected at various time intervals (0, 1, 3, 6, and 12 h) for gene expression analysis. Seedlings moved to normal MS medium were used as the reference. Each stress treatment was repeated three times for precision. The qRT-PCR primer details can be found in Supplementary Table S1.

4.4. Promoter Cis-eElement Analysis

The 2000 bp promoter sequences upstream of the start codon of StPYL16 were extracted using TBtools. Subsequently, the sequence was analyzed for cis-acting elements using the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 23 May 2024). Next, the StPYL16 promoter was inserted into the plant expression vector pBI101-GUS through homologous recombination. Tobacco leaves were transiently transformed with Agrobacterium tumefaciens GV3101, and GUS staining was conducted after 2 days to assess promoter activity. Meanwhile, tobacco plants with the StPYL16 promoter were exposed to stress by transferring them to Hoagland solution containing 200 mM mannitol, with control plants growing in normal Hoagland solution. Additionally, hormone treatment involved spraying with 100 mM ABA, while another group was sprayed with alcohol as a control. After 6 h of treatment, samples were collected for GUS histochemical staining.

4.5. Evaluation of Drought Resistance after Transient Transformation of Tobacco

The coding sequence (CDS) of StPYL16 was obtained through PCR amplification using specific primers (Table S1). The PCR product was then digested with Bgl Ⅱ and Spe Ⅰ before being inserted into the pCAMBIA1304 vector to generate the pC1304-StPYL16 construct. This construct was subsequently introduced into Agrobacterium tumefaciens GV3101 using the heat shock method for further experiments. To investigate the function of StPYL16, tobacco plants were transferred from normal growth conditions to Hoagland nutrient solution after approximately 20 d of growth and cultured for 2 d. Following this, tobacco leaves were infiltrated with GV3101 carrying pC1304-StPYL. The empty vector pC1304 was used as a negative control. After 2 d of normal growth, both the experimental and control samples were exposed to stress by transferring the entire plants into Hoagland nutrient solution supplemented with 200 mM mannitol. Samples were collected after 6 h for GUS staining to confirm successful transformation. Additionally, various physiological parameters related to stress response were also evaluated.

4.6. Evaluation of Drought Resistance after Stable Transformation of Tobacco

The recombinant plasmid pC1304-StPYL16 was used for transformation of Nicotiana tabacum (T12) via the Agrobacterium-mediated leaf disk method. The transgenic plants were then cultured on Murashige and Skoog (MS) medium and screened with 1/2 MS medium containing kanamycin (50 mg L⁻¹, w/v) for selection. Positive lines were identified and confirmed through qRT-PCR and GUS staining. From the qRT-PCR results, three transgenic lines showing the highest expression of StPYL16 were selected. These lines were then cut into uniform stem segments and subjected to stress by inoculation on MS medium supplemented with 100 mM, 200 mM, and 300 mM mannitol, while normal MS medium was used as the control. After a 30-day stress period, the physiological indicators related to stress were evaluated.

4.7. β-Glucuronidase (GUS) Staining

Histochemical staining for GUS activity was performed as described previously [50]. The transgenic plant leaves were harvested and immersed in the reaction solution (2 mM 5-bro-mo-4-chloro-3-indolyl-β-d-glucuronic-acid, 50 mM sodium phosphate, 10 mM EDTA, 2 mM ferricyanide, and 0.1% Triton X-100, pH 7.0) and incubated at 37 °C for 12 h under dark conditions. Subsequently, the stained leaves underwent gradient elution with 30%, 60%, and 100% ethanol in an 80 °C water bath. Following the complete disappearance of chlorophyll, photomicrographs were captured using an optical microscope.

4.8. Determination of Phenotypic and Physiological Indicators

The proline and MDA contents were determined according to a previous publication [51]. The enzymatic activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in both the transgenic lines and control plants were measured using commercial assay kits (BC0170, BC0220, and BC0200; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) with spectrophotometric methods. Phenotypic traits primarily consisted of plant height, stem diameter, and root length. Additionally, a root scanner (LD-WinRHIZO) was employed to assess parameters such as total root length, number of root tips, root area, and root volume of the plant root system.

4.9. Expression Measurement of Stress-rResponsive Genes

Total RNA was extracted from a tobacco plant and reverse transcribed to synthesize cDNA. The expression of stress-related genes was assessed by qRT-PCR using specific primers (Table S1).

4.10. Statistical Analysis

Three plants were sampled for each of the three biological replicates in every treatment. Mean and standard deviation (SD) were calculated for each treatment based on data collected from these replicates. The variance between the three overexpression (OE) lines and control plants was evaluated using one-way analysis of variance (ANOVA) in SPSS v19.0 (SPSS Inc., Chicago, IL, USA), with statistical significance set at p < 0.05 or p < 0.01.

5. Conclusions

In conclusion, this research unveiled the significance of the potato ABA receptor gene StPYL16 in the plant’s response to drought stress. Moreover, the over-expression of StPYL16 has been shown to boost plant performance under drought conditions. Significantly, our study offers a valuable approach for genetically modifying future crops to adapt to progressively adverse environments.