Introduction

Water is a critical component in ensuring the viability and sustainability of life across various living organisms. In plants, drought can be characterized as a physiological condition resulting from inadequate soil hydration, leading to a deficiency in water availability for photosynthetic organisms, which adversely influences their biological processes1,2,3. Drought stress causes the formation of reactive oxygen species (ROS), consisting of free radicals, including hydroxyl and superoxide radicals, or non-radicals such as singlet oxygen and hydrogen peroxide4. The ROS production can result in oxidative damage to essential molecules such as proteins, membrane lipids, and DNA, potentially disrupting normal plant cell functions5. When plants face oxidative stress, they initiate different molecular, biochemical, physiological, and morphological changes mediated by ROS signaling6,7.

Numerous studies have been dedicated to investigating both ROS generation and collecting mechanisms in plants, alongside the roles of these mechanisms throughout plant development and responding to either abiotic or biotic stresses8,9. To mitigate oxidative damage within plant cells, the presence of protective enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), is fundamental in maintaining ROS homeostasis10. Currently, investigations have predominantly concentrated on the function of SOD as the initial enzyme involved in the enzymatic antioxidant defense pathway11.

The SODs, as widespread metalloenzymes, facilitate the conversion of superoxide radicals into hydrogen peroxide (H2O2) and molecular oxygen (O2). Given the potential of superoxide radicals to evolve into highly reactive hydroxyl radicals, SODs significantly contribute to plants defense mechanisms against oxidative stress12,13. These enzymes are classified into three categories based on the metal cofactors they contain: iron SODs (FeSOD), copper/zinc SODs (Cu/ZnSOD), and manganese SODs (MnSOD)14. Inside plant cells, different isoforms of these enzymes, including FeSOD, MnSOD, and multiple variants of Cu/ZnSOD, are nuclear-encoded and distributed across various subcellular compartments, each distinguished by its specific metal cofactor15. It has shown that their functions are key components of the plant’s abiotic stress tolerance machinery. The Cu/ZnSOD has attracted more investigative focus regarding its function in abiotic stress tolerance than the MnSOD16. Among the different isoforms of superoxide dismutase, Cu/ZnSOD plays a broader role in regulating oxidative balance in plants due to its diverse localization in the cytosol, chloroplasts, and peroxisomes. While MnSOD is mainly active in mitochondria and FeSOD is found primarily in chloroplasts, Cu/ZnSOD occurs in multiple organelles and serves as the first line of defense against superoxide accumulation in the cell17. Moreover, Cu/ZnSOD genes usually exhibit higher sensitivity to abiotic stresses such as salinity, drought, and temperature, and their expression often increases during the early stages of stress response. Several studies have shown that overexpression of Cu/ZnSOD genes in different plant species enhances stress tolerance and reduces oxidative damage18.

Drought conditions stimulate activating genes engaged in stress, fostering the accumulation of metabolically functional compounds and initiating the biosynthesis of particular proteins19. Plants employ intricate mechanisms to alleviate the detrimental effects associated with drought conditions20. A large number of genes that responded to drought stress have been found, cloned, and subsequently applied as potential candidates in the field of genetic engineering21. For instance, genes encoding proteins critical for abiotic stress resilience include antioxidative enzymes (SOD, CAT, APX, GPX, DHAR and GR), enzymes necessary for osmolyte biosynthesis (Protein kinases, Amino acid synthases, Glucose-6-phosphate dehydrogenase, Sorbitol dehydrogenase and Phenylalanine ammonia-lyase), and enzymes that play a role in regulating and transcribing genes responsible for stress (RNA polymerase, Histone acetyltransferases (HATs), Histone deacetylases (HDACs), DNA methyltransferases, Protein kinases (e.g., MAPK), Chromatin remodeling enzyme and Transcription factors (e.g., WRKY, CBF, MYB, bHLH, ERF)22,23,24,25,26. The regulatory pathway for drought stress tolerance in plants is illustrated in Fig. 1.

Fig. 1
Fig. 1
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Schematic representation of plant signaling networks and physiological responses to drought stress. Drought stress activates reactive oxygen species (ROS), hormonal signaling (ABA, JA, SA), calcium, and osmotic stress signaling pathways, which lead to the activation of protein kinases (CDPKs, MAPKs). These pathways regulate transcription factors (WRKY, NAC, DREB, MYB ERF), which in turn modulate the activities of antioxidant enzymes (SOD, CAT, APX, GPX), promote osmolyte accumulation (proline, glycine betaine), and control stomatal regulation and ion homeostasis, thereby collectively enhancing drought tolerance and maintaining plant homeostasis.

The initial physiological adaptation of plants under drought stress primarily involves stomatal closure to reduce transpiration, serving as a crucial strategy to minimize water loss27,28. However, this response concurrently limits CO2 uptake, thereby decreasing photosynthetic efficiency29,30. Reduced transpiration further restricts nutrient uptake and translocation from roots to aerial tissues, leading to ionic imbalance within plant cells31,32. These disruptions adversely affect various physiological processes, particularly photosynthesis, which is directly associated with plant growth and productivity33,34. Moreover, drought stress impairs chloroplast integrity and chlorophyll biosynthesis, resulting in decreased photosynthetic capacity and the onset of oxidative stress within plant tissues34,35.

Enhancing drought tolerance through transgenic approaches has emerged as an effective strategy to improve plant resilience. Overexpression of SOD genes in transgenic plants has been shown to increase enzymatic activity, promote ROS scavenging, and mitigate oxidative stress, particularly in the context of ROS signaling within chloroplasts36. Milk thistle is recognized as a drought-tolerant species, and its unique characteristics in withstanding environmental stress make it a promising donor species for genetic engineering to enhance drought tolerance in other plants.

In this study, we selected the SmSOD gene from Silybum marianum due to its high antioxidant activity and reported role in drought tolerance in its native plant. Tobacco was chosen as the host species because of its economic importance and sensitivity to drought, making it a suitable model for studying stress responses.

The aim of this study was to investigate the effect of SmSOD overexpression on drought tolerance in Tobacco. We systematically assessed physiological parameters (photosynthetic efficiency and stomatal morphology), enzymatic activity (SOD activity), and transcriptional level (SmSOD expression) under both control and drought stress conditions to provide a comprehensive understanding of SmSOD-mediated stress adaptation.

Materials and methods

Experimental design and sampling

The experiment was performed at the experimental field of Shahid Beheshti University, Tehran, Iran (51.23° N and 35.48° E). The soil of the experimental site was classified as loam with a pH of 7.27 and an electrical conductivity (EC) of 2.13 dS/m. The soil contained 29.5% clay, 35% silt, and 44% sand, with a field capacity of 39% and available water content of 33%. The experimental design and related details have been previously described comprehensively37. To isolate the target SmSOD gene, Milk thistle plants cultivated under field conditions while under severe drought stress (40% field capacity (FC)) were used. Sampling began eight days after the application of the stress condition. For this purpose, three plants were randomly selected, and medium-sized leaves in the middle parts of the stem were collected. The leaves were placed in liquid nitrogen, and thereafter kept at − 80 °C until further analysis.

Primer design and isolation of SmSOD gene from milk Thistle

Since the SmSOD gene was not identified in Milk thistle and its sequence was unavailable on the NCBI website, the sequence of this gene was identified in plants from the same family as Milk thistle, such as Sunflower and Safflower. Subsequently, these sequences were subjected to BLAST38 on the NCBI site to identify similar sequences. The obtained gene sequences were then stored and aligned using the T-COFFEE Multiple Sequence Alignment Server39. To design primers, conserved regions were used at the start and end of the gene sequence, encompassing the start and stop codons. Due to the unknown sequence of this gene in Milk thistle, the primers were designed degenerately from the total obtained sequences.

Vector NTI40 and Oligo741 software were employed for the evaluating parameters such as melting temperatures (TM), GC content, stem-loop structures, homodimer, and heterodimer of primers, which were subsequently synthesized by Bioneer Company (South Korea). The details of the primers for SmSOD and housekeeping genes, including sequences and other characteristics, are presented in Table 1. To extract RNA, 0.2 g of fine powdered fresh leaves (using liquid nitrogen) were used employing the RNX Plus kit (RB429A, Sinaclon, Iran). Then, RNA samples were treated with DNase I (RB125A, RNA, Iran) to remove any potential genomic contamination. The synthesis of cDNA was carried out in a 20 µL reaction volume using reagents from a cDNA synthesis kit (RB225A, Parstous, Iran), following the provided manufacturer’s protocol, and synthesized cDNA was subsequently kept at − 20 °C until further analysis.

The PCR was conducted on the target gene and housekeeping genes using specific primers, as listed in Table 1. PCR mixtures included 10 µL of Master Mix (RNA, Iran), 1 µL of each forward and reverse primer,1 µL of cDNA, and 7 µL water. The thermal cycler was programmed for an initial denaturation phase at 94 °C for 5 min, succeeded by 35 cycles at 94 °C for 20 s, 57 °C for 15 s, and 72 °C for 20 s, with a final extension period for 5 min at 72 °C. After the amplification, the PCR products were assessed through electrophoresis on a 1% agarose gel. After electrophoresis, the DNA fragments were carefully cut out from the gel using a sterile scalpel and placed into a 1.5 mL microtube. DNA purification was done using the AccuPrep® Gel Purification Kit (Bioneer Pacific, K-3038, Germany), adhering to the manufacturer’s protocol. Successful purification was verified through a subsequent round of PCR on the purified solution.

Table 1 Characteristics of primers used in this experiment.
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Construction of Recombinant vector (pTG19- SmSOD)

The purified product was inserted into the pTG19 plasmid vector (Sinaclon, PR911643, Iran) according to the manufacturer’s guidelines. Subsequently, the plasmid, once ligated, transformed into a competent Escherichia coli (E. coli) DH5α strain using the thermal shock method42. This was followed by incubation in Luria-Bertani (LB) broth medium without antibiotics for one hour at 37 °C. Blue-white screening, PCR, enzymatic digestion, and sequencing were employed to verify the gene’s successful cloning. Subsequently, the plasmid was isolated from the bacteria constituting the white colonies using the GTP Plasmid DNA Extraction Kit (Gene Transfer Pioneers, IRAN), followed by digestion with the BamHI enzyme. These reactions were incubated at 37 °C for 16 h. The products obtained were examined through electrophoresis (1% agarose gel), and their sizes were compared against a 1 kb DNA ladder (SL7052, Sinaclon, IRAN).

Sequencing, assembling, and gene submission to the NCBI website

The plasmid pTG19, containing the SmSOD gene, was sent to Bioneer company (South Korea) for Sanger dideoxy sequencing in both directions (\(5^{\prime}\to{3}^{{\prime}}\) and \(3^{\prime}\to{5}^{{\prime}}\)) with M13 primers (forward and reverse). The length of reads was 1000 bp in each direction. The forward and reverse sequences obtained from sequencing were assembled by Vector NTI Advance® 11.0 and ChromasPro 2.0 software43. Then, the resulting sequence was subjected to BLAST at the NCBI to confirm the accuracy of the sequence. To submit the SmSOD gene sequence to the NCBI database, the coding sequences (CDS) of selected sequences derived from Milk thistle were first extracted by Vector NTI software, and then the resulting sequence was submitted to NCBI following the site’s submission guidelines.

Construction of expression Recombinant vector (pBI121 + SmSOD)

In this study, the plasmids pTG19 + SmSOD (recombinant) and pBI121, serving as the expression vector, were subjected to enzymatic digestion using BamHI enzymes. This procedure was succeeded by the purification of the digested products—the pBI121 plasmid and the SmSOD fragment—employing the AccuPrep® Gel Purification Kit. The SmSOD fragment underwent ligation into the BamHI-digested pBI121 plasmid, according to Sambrook and Russell’s44 method. Then, the ligated product was introduced into E. coli DH5α strain through standard transformation protocols. The culture of the transformed E. coli was performed in LB broth medium without antibiotics using a shaker incubator at 37 °C for one hour. The transformed bacteria were spread onto LB agar plates supplemented with kanamycin at 50 mg/L and incubated at 37 °C for 16 h. To verify the incorporation of the recombinant pBI121 plasmid, PCR amplification and restriction digestion using the EcoRI and HindIII enzymes were performed45,46.

Conformation of Agrobacterium tumefaciens LBA4404 and tobacco inoculation

The transformation of the recombinant expression plasmid into A. tumefaciens was accomplished using the freeze-thaw method described by Höfgen and Willmitzer47. Moreover, A. tumefaciens strain LBA4404 facilitated the genetic modification of Tobacco (Nicotiana xanthi) via an adapted leaf disc method48. Following sterilization using a 0.02% HgCl2 solution for 12 min, Tobacco seeds were subsequently cultured in Murashige and Skoog (MS) culture medium without any hormones. After a growth period of 10 days, the resulting plantlets were separated and sliced into smaller fragments using a scalpel. These fragments were then prepared for bacterial inoculation procedures49.

Three days before the inoculation with A. tumefaciens, a pre-culture of leaf disc explants using the MS medium was done. Following the pre-culture period, A. tumefaciens (LBA4404 with OD600 = 0.7) was used to inoculate these wounded explants for 7 min. The explants were dried using filter paper and transferred to a co-culture medium. This medium was enriched with 0.2 mg/L of Indole-3-acetic acid (IAA) and 2.5 mg/L of 6-Benzylaminopurine (BAP) to facilitate direct somatic embryogenesis, as well as 1 mg/L of BAP and 0.1 mg/L of 1-Naphthaleneacetic acid (NAA) aimed at direct shoot induction for 3 days. Afterward, the explants were placed onto a selection medium that consisted of a full MS supplemented with 1 mg/L of BAP and 0.1 mg/L of NAA for direct shoot induction as well as 50 mg/L of kanamycin and 150 mg/L of cefotaxime. Subculture of the explants was carried out every 10 days. Root induction of the explants was performed using a half MS medium, which included 0.1 mg/L of NAA. The growth chamber maintained at 25 °C with a photoperiod of 16 h of light and 8 h of darkness, was used for all tissue culture experiments. Putative transgenic plants that demonstrated appropriate growth characteristics were used for subsequent experimental analysis50. Specific primers were used to perform PCR on the putative transgenic plants. This process started with grinding fresh leaf and shoot tissues obtained from putative transgenic lines using liquid nitrogen. The genomic DNA was extracted using the Dellaporta et al.51 protocol. The PCR targeted the SmSOD, 18SrRNA, and NPTII genes to ascertain the transgenic status of the lines under investigation. To validate the absence of contamination and establish negative and positive controls, water samples (PCR mixture without DNA template), wild-type plant material, and a recombinant plasmid were used, respectively. A total of 100 leaf disc explants were inoculated, from which 20 transgenic plants were generated.

Tobacco plant cultivation and drought application

Initially, transgenic and control tobacco plants were grown in a controlled laboratory environment (phytotron). Once the plants reached the four-leaf stage, they were transferred to pots containing perlite and covered with a plastic sheet, which was misted with water daily. To facilitate acclimatization, small valves were made in the plastic cover, which was then gradually removed while maintaining a regular watering schedule.

Subsequently, the plants were transferred to larger pots filled with a mixture of leaf mold, perlite, and sand. At this stage, sparing irrigation was replaced with regular watering, and the plants were gradually transitioned from the phytotron to the laboratory and then to the greenhouse.

After full acclimatization, at the eight-leaf stage, the plants were subjected to drought stress treatments. For this purpose, a total of 20 plants were used, consisting of 10 transgenic plants and 10 control plants. Of each group, 5 plants were subjected to drought stress (irrigated at five-day intervals: ~50% Field Capacity), and 5 plants were watered regularly. Finally, the plants were used for various downstream analyses; three biological replicates per treatment were selected, and sample collection was carried out according to the requirements of each assay.

Molecular and physiological characteristics

Gene expression analysis

In this study, RNA was extracted from 0.2 g of fresh Tobacco leaves using an RNA extraction kit (RB1001, RNA, Iran), followed by DNase I (RB125A, RNA, Iran) treatment to eliminate probable genomic contamination. The quantity and quality of isolated RNA were evaluated using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA) and electrophoresis in a 1% agarose gel. Extracted RNA served as a template for synthesizing the cDNA, utilizing a cDNA synthesis kit (RB125A, RNA, Iran) according to manufacturer guidelines. Then, the cDNA was stored at -20 °C until further analysis. Primers were designed by considering regions proximal to the polyadenine tail, covering 150 to 250 bp for qPCR. Parameters including the Tm, GC content, stem-loop structures, homodimer, and heterodimer formation were assessed utilizing Oligo software (version 7.60) and Vector NTI® Express Designer Software (version 11). The reference gene was 18SrRNA (GenBank ID: OR083346). Table 1 lists primer sequences along with additional details are listed.

RT-qPCR amplifications were carried out employing the Rotor-Gene 2000 apparatus (Corbett Life Science, Australia) by utilization of SYBR® Green Real-Time PCR Master Mix (RB120, RNA, Iran). A total volume of 20 µL was used to prepare the reactions, which included 10 µL of the 2x SYBR solution, 1 µL of cDNA, 1 µL of each primer at a concentration of 20 nmol, and 7 µL of RNase-free water. The thermal cycling program began with a 5-min initial denaturation phase at 95 °C, succeeded by 35 amplification cycles (1 min of denaturation at 95 °C, 15 s of annealing at 57 °C, and 20 s of extension at 72 °C). Specificity verification for each amplicon was obtained by evaluating post-amplification melting curves, ranging between 60 and 95 °C. The cycle threshold (Ct) values, as well as primer efficiency, were obtained using the LinRegPCR software (11.0)52. Moreover, the calculation for relative expression levels was conducted using the Relative Expression Software Tool (REST) (2009), according to the Pfaffl method53, incorporating the efficiency of the primers (E) in the following formula, as described by Livak et al.54:

$${\text{Gene}}\;{\text{expression}}\;{\text{ration}} = \left( {{\text{E}}_{{{\text{Gene}}}} } \right)^{{\Delta ct\;{\text{Gene}}}} /\left( {{\text{E}}_{{{\text{Ref}}}} } \right)^{{\Delta ct\;{\text{Ref}}}}$$

Three biological and three technical replicates were used to perform all qRT-PCR analyses to ensure the reliability and reproducibility of data.

Chlorophyll fluorescence measurement

Fully developed young leaves were used in this experiment to measure parameters obtained from chlorophyll fluorescence imaging in both normal and stress-induced plants. While still attached to their respective plants, these leaves underwent a 20-min dark adaptation. Following this, their slow chlorophyll fluorescence induction was slowly measured using a FluorCam (FluorCam FC 1000-H, Photon Systems Instruments, PSI, Czech Republic) equipped with a CCD camera and four fixed LED panels, which provided measuring pulses and induced saturating flashes. The maximum quantum yield of photosystem II (Fv/Fm) was calculated using a specialized method55,56. Chlorophyll fluorescence measurements began during exposure to short flashes in darkness, followed by a saturating pulse (3900 PPFD) at the end, which stopped the electron transport by reducing quinone acceptors57. The protocol involved capturing two sets of fluorescence readings: one during exposure to the saturating flash (Fm) and the other averaged throughout short flashes in darkness (F0). The calculation of Fv/Fm was performed employing the following equation:

$${\text{F}}_{{\text{v}}} /{\text{F}}_{{\text{m}}} = \left( {{\text{F}}_{{\text{m}}} - {\text{F}}_{0} } \right)/{\text{F}}_{{\text{m}}}.$$

To quantify non-photochemical quenching (NPQ), the maximum fluorescence in light-adapted steady state \(\left({F}_{m}^{\prime}\right)\) Wasw also taken. Using this measurement, the NPQ was determined based on the following equation58:

$$\text{NPQ} = ({\text{F }}_{\rm m}/{\text{ F}}_{\rm m}^{\prime} )-1$$

The data was calculated using FluorCam software (version 7) (PSI, Czech Republic).

Measurement of stomata morphological parameters

The fully developed young leaves from both the control group and those subjected to drought stress treatments were selected for stomatal length, width, index, diversity, and pore width measurements, according to Aliniaeifard and van Meeteren’s59 method. For microscopic analysis, segments of the leaves, positioned equally from the leaf’s edges and located halfway between the apex and the base, were selected. A nail polish was used to cover the lower epidermis of fully developed young leaves. After 10 min, the dried polish was removed using transparent sticky tape. Subsequently, samples of the dried polish adhered to the sticky tape were affixed to microscope slides to examine stomatal morphological characteristics under a light microscope.

Stomatal parameters were quantitatively assessed utilizing the Omax top-view software (version 3.5), followed by a detailed analysis with ImageJ software (U.S. National Institutes of Health, Bethesda, MD) (1.8.0). This evaluation included stomatal width and length, pore width and length, and stomatal density (SD) measurements. Fifty stomata from leaves subjected to each specific treatment were investigated. Stomatal density is defined as the frequency of stomata per unit of leaf surface area60. Moreover, the analysis included calculating the stomatal index, a metric denoting the ratio of stomatal pores to the total number of epidermal cells (ED), established as a percentage61. The stomatal index was calculated following an equation presented by Smith et al.62, incorporating SD and the density of ED cells per unit area of the leaf surface.

$${\text{Stomatal}}\;{\text{index}} (\%) = \left( {{\text{SD}}/{\text{SD}}} + {\text{ED}} \right) \times 100.$$

Determination of SmSOD enzymatic activity

At first, 1 g of leaves samples was subjected to liquid nitrogen and ground into 10 mL of extraction buffer comprised of 0.1 M phosphate buffer at pH 7.5, supplemented with 0.5 mM EDTA. Next, the homogenate was filtered through a quadruple cheesecloth layer and centrifuged at 15,000×g for 20 min. All procedural steps dedicated to the extraction of enzymes were conducted at 4 °C to ensure enzymatic integrity. The assessment of total SOD activity was carried out by measuring the enzyme-mediated suppression of nitro blue tetrazolium (NBT) reduction. This was performed by adding two mM riboflavin (0.1mL) into the reaction mixture (3 mL), which consisted of 0.1 mM EDTA, 13.33 mM methionine, 75 µM NBT, and 50 mM phosphate buffer (pH 7.8), 50 mM sodium carbonate, and 0.1mL of the enzyme extract. Subsequent exposure of the mixture to two 15 W fluorescent lamps for 15 min initiated the reaction. Quantitative absorbance measurement was done at a wavelength of 560 nm, with one unit of enzyme activity defined as the requisite amount of enzyme that obtains a 50% reduction in absorbance compared with control tubes without the enzyme63.

Statistical analysis

All molecular and physiological data were analyzed using R software version 3.5.364. Across all analyses, each treatment included three biological replicates, and each biological replicate was measured in three technical replicates. Data normality was verified using the Shapiro–Wilk test from the stats package. ANOVA was performed on the data for a completely randomized design (CRD), with treatments considered as fixed effects and replicates as random effects. Furthermore, Duncan’s multiple range test was used for mean comparisons, utilizing the agricolae package, with a significance level of 5%65. All figures and tables were generated using Microsoft Word and Excel 2013.

Results

Cloning of SmSOD gene into pTG19 and pBI121 vector

The SmSOD gene fragment was cloned into the pTG19 cloning vector to generate more gene copies and create sticky ends for ligation with digestion sites. After 16 h of incubation at 37 °C, white colonies appeared on the E. coli plates, indicating successful cloning (Fig. S1a). To verify whether the SmSOD gene is present in these colonies, a digestion with the BamHI enzyme was conducted. The gel electrophoresis of the digestion product validated that the SmSOD gene is present in the pTG19 vector. The pTG19 vector map indicates a sequence length of 2880 bp, which increased to 3342 bp after inserting the SmSOD gene. Following digestion with the BamHI enzyme, the fragment lengths were 462 bp and 2880 bp, as confirmed by the gel photo (Fig. S2a). To validate the correctness of the SmSOD gene, the vector containing the gene was sequenced, and after assembly, a BLAST search on the resulting gene sequence was performed. BLAST analysis of the obtained sequence demonstrated high similarity with SmSOD genes from related plant species, confirming that the cloned gene encodes a Cu/Zn-superoxide dismutase (Cu/Zn-SOD). Subsequently, the 462 bp sequence of the SmSOD gene isolated from Milk thistle was submitted to the NCBI site with the accession number MG893090.

Fig. 2
Fig. 2
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Schematic representation of the pBI121 vector with the SmSOD gene inserted. The diagram shows the key elements of the plasmid, including the CaMV 35 S promoter (for driving gene expression), the SmSOD gene (inserted between the BamHI sites), the NOS terminator, and the NeoR/KanR marker for selection. It also depicts the LB T-DNA repeat, RB T-DNA repeat, and restriction enzyme sites (BamHI, HindIII, EcoRI) used for cloning. The total length of the plasmid is 13,414 base pairs.

After successfully isolating the SmSOD gene, its sequence was digested from the pTG19 vector using the BamHI enzyme and afterward inserted into the pBI121 expression vector. This pBI121 vector was designed to express the carried gene in plant cells. Similar to the pTG19 vector, cloning results with pBI121 also appeared as white colonies on the plate (Fig. S1b). Subsequently, digestion was performed with HindIII and EcoRI enzymes. According to the vector map pBI121 + SmSOD (Fig. 2), the expected fragment lengths are 1680 bp and 11,734 bp (Fig. S2b). Additionally, to further confirm the cloning, the gene carrier vector was digested with the BamHI enzyme, resulting in fragments of 462 bp and 12,952 bp (Fig. S2c).

Plant inoculation with gene-carrying bacteria and Transgenic plant development

The expression vector was introduced into Agrobacterium tumefaciens to deliver the SmSOD gene into Tobacco plant cells via inoculation. Subsequently, plant tissues were cultured in a selective MS medium containing Kanamycin and Cefotaxime. Tissues lacking the transgene wilted and turned brown, whereas tissues containing the transgene proliferated successfully in the selective medium (Fig. 3a). Wild-type plants were also cultured under the same MS conditions; however, since they did not carry the transgene, no selection with antibiotics was applied. The seedlings developed roots and shoots (Fig. 3b) and were then transferred to pots and a controlled laboratory environment for acclimatization (Fig. 3c). These acclimated seedlings were subsequently subjected to drought stress (Fig. 3d). In Fig. 3, wild-type samples are also indicated, allowing a clear visual comparison with transgenic plants.

Fig. 3
Fig. 3
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The process of transformation and application of drought stress in tobacco plant. a Selection of transgenic plants in selective medium containing antibiotics. b Root and shoot induction by hormones in transgenic plants. c Adaptation of transgenic plants to soil media in laboratory conditions. d Application of drought stress to adapted tobacco seedlings.

Confirmation of transgenicity of tobacco seedlings by PCR

To confirm the transgenic status of the tobacco seedlings, PCR was performed using two specific primer pairs targeting the SmSOD and NPTII genes. Prior to using these primers, PCR was performed on wild-type (WT) tobacco plants, and no amplification was observed, confirming that the primers do not amplify endogenous SOD genes. Gel electrophoresis revealed two distinct bands, measuring 462 bp and 350 bp, corresponding to the SmSOD and NPTII sequences, respectively, thereby confirming the transgenic nature of the analyzed seedlings (Fig. S2c).

Effect of the SmSOD gene on drought stress tolerance in Transgenic plants

Gene expression analysis by qRT-PCR

The comparative expression of the SmSOD target gene, using 18 S rRNA as a reference, was analyzed on both transgenic and wild-type Tobacco plants exposed to water scarcity stress, and the results were compared with the plants under control conditions. The level of SmSOD expression in transgenic plants subjected to drought stress significantly increased (17.012-fold increase) at a 0.01 significance level. Moreover, when evaluating drought stress tolerance, our results demonstrated that the SmSOD expression in transgenic plants was almost five times higher than in wild-type counterparts (Table 2).

Table 2 Gene expression analysis for SmSOD and 18SrRNA in control and drought stress conditions. Reaction efficiency values represent the efficiency of the PCR reactions, and expression values are normalized to the 18SrRNA reference gene. The p-value indicates statistical significance, with thresholds of p < 0.01. A denotes that the SmSOD-T sample group is significantly different from the control group (p < 0.01). UP indicates upregulation of SmSOD expression under drought stress conditions.
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SOD enzyme activity

The enzymatic activity of SOD in transgenic plants exposed to water scarcity conditions was significantly increased (9.5 ± 0.4 Units/mg protein) compared with its activity in the control group (4.5 ± 0.2 Units/mg protein). In wild-type plants, the enzyme activity under drought stress rose to 5.2 ± 0.3 Units/mg protein, compared with 3.3 Units/mg protein in their control group. This represented an 82.6% increase in SOD enzyme activity in transgenic plants subjected to water deficiency compared with the wild-type plants. The highest to lowest observed SOD enzymatic activities were for drought-subjected transgenic plants, drought-subjected wild-type plants, control transgenic plants, and control wild-type plants, respectively (Fig. 4).

Fig. 4
Fig. 4
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SOD enzyme activity in transgenic tobacco plants under drought stress. Different letters above bars indicate significant differences at p < 0.05, as determined by Duncan’s multiple range test. Error bars represent standard error (SE).

Photosynthetic functionality influenced by SmSOD transferring to tobacco plants

To assess how the SmSOD gene affects the resilience of transgenic plants to stress, an investigation of the plant’s photosynthetic apparatus functionality was performed. This study focused on two critical features related to the Fv/Fm and the protective stress mechanism: Non-photochemical quenching light steady-state (NPQ.lss) and Maximum Quantum Yield (QYmax). A significant increase (p < 0.05) in NPQ levels was observed in both transgenic and wild-type plants exposed to water scarcity conditions compared with their levels in the control group (Fig. 5a). The average NPQ in transgenic plants under water deficiency conditions was 0.426 ± 0.04, while in non-stressed transgenic plants, it was 0.193 ± 0.01. In wild-type plants, the average NPQ under water scarcity conditions was 0.363 ± 0.03 and 0.173 ± 0.01 in non-stressed conditions. The percentage increase in the NPQ under drought stress was 120.72% for transgenic plants and 109.82% for wild-type plants. Additionally, transgenic plants exposed to water scarcity stress showed 17.3% higher NPQ than wild-type plants. Transgenic plants had 11.5% higher NPQ in control conditions than the wild-type group.

Parallel to the NPQ observations, a significant increase (p < 0.05) in QYmax (also known as the value of Fv/Fm) values was observed in both transgenic and wild-type plants under water deficiency conditions compared with controls. The difference in Fv/Fm QYmax values under water scarcity conditions compared with control conditions was recorded at 0.27 ± 0.01 for transgenic plants and 0.22 ± 0.01 for wild-type plants. The increase in QYmax attributable to drought stress was calculated at 42.65% for transgenic plants and 35.31% for wild-type plants. In a comparative analysis under water scarcity conditions, transgenic plants had a 7.11% higher Fv/Fm QYmax than wild-type plants. Additionally, there was a slight increase of 1.60% in Fv/Fm QYmax in control group plants (Fig. 5b).

Fig. 5
Fig. 5
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a Non-photochemical quenching (NPQ) and b Maximum photosystem II quantum efficiency derived from chlorophyll fluorescence parameters exhibited by leaves of tobacco plants in response to drought stress. Statistical significance is indicated at p < 0.05, as determined by Duncan’s multiple range test, and error bars represent the standard error (SE).

Fv/Fm was significantly compromised in wild-type plants under water scarcity stress. In contrast, transgenic plants exposed to the same conditions showed a lesser degree of reduction (Fig. 6). The Fv/Fm ratio in wild-type plants showed a significantly greater decrease under drought stress compared with the Fv/Fm ratio in transgenic plants exposed to the same conditions. In the evaluation of Fv/Fm imaging, plants subjected to drought stress typically showed a higher Fv/Fm (brighter yellow hue), in contrast to control plants, which present a redder coloration (Based on the scale provided close to warmer color indicate higher Fv/Fm and close to cooler color indicate lower Fv/Fm.

Fig. 6
Fig. 6
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Representative images of maximum quantum yield of photosystem II (Fv/Fm) obtained from the leaves of tobacco plants in response to drought stress. The color scale in the bottom of the image represents the level of Fv/Fm. Close to warmer color (close to 1) indicates higher Fv/Fm and close to cooler colors indicates lower Fv/Fm.

Stomatal morphology influenced by SmSOD transformation

The images prepared for apertures in four samples (Drought stress-wild plants, Drought stress-transgenic plants, Control- Wild plants, Control- Transgenic plants) are presented in Fig. 7. Observations revealed that plants subjected to drought stress had a lower stomatal density than those grown under control conditions (Fig. 8a). The stomatal density measured for transgenic and wild-type plants was 24.5 and 18.5 stomata per mm2, respectively, under control conditions. However, under drought stress, the stomatal density in transgenic and wild-type plants decreased to 15.5 and 11.5 stomata per mm2, respectively.

Fig. 7
Fig. 7
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Stomatal anatomy on transgenic tobacco leaves from plants grown under drought stress. The scale bar unit is in nanometers (um). SEM images at 10,000X magnification.

The percentage decrease in stomatal density was 58% for transgenic plants and 60.8% for wild plants. In contrast to the pattern observed in stomatal density, the width of stomata in both transgenic and wild-type plants increased under drought conditions. In transgenic plants, the stomatal width expanded from 0.31 ± 0.02 μm to 0.25 ± 0.01 μm under drought stress, while in wild-type plants, it decreased from 0.26 ± 0.01 μm to 0.22 ± 0.01 μm. The decrease in stomatal width under drought stress was 24% for transgenic plants and 18.1% for wild-type plants. Notably, under drought conditions, the stomatal width of transgenic plants was 19.2% smaller than that of wild-type plants (Fig. 8b). Furthermore, the analysis of stomatal length in both transgenic and wild-type plants subjected to either drought or control conditions revealed no significant differences, with measurements ranging between 0.3 ± 0.02 μm and 0.5 ± 0.03 μm.

Fig. 8
Fig. 8
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Stomatal characteristics of wild-type and transgenic tobacco plants under control and drought stress conditions. a Stomatal density (stomata per mm2). b Stomatal width (µm). Different letters above bars indicate significant differences at p < 0.05, as determined by Duncan’s multiple range test. Error bars represent standard error (SE).

Discussion

This study compared transgenic and wild-type plants subjected to drought stress and under control conditions. Three key parameters, including SmSOD gene expression, SOD enzyme activity, plant photosynthetic system functionality, and stomatal morphology in response to drought stress, will be discussed.

SmSOD gene expression and enzyme activity

SOD catalyzes the conversion of superoxide into hydrogen peroxide and oxygen, making it an essential component of the plant antioxidant defense system66. The results of this study showed that in transgenic plants under drought stress (SmSOD -T), SmSOD expression was significantly upregulated, reaching 17.012-fold relative to the control (P = 0.001). In contrast, wild-type plants under drought stress (SmSOD -W) showed no significant change (3.373-fold, P = 0.240). Moreover, the comparison between transgenic and wild-type plants under drought stress revealed that SmSOD expression in transgenic plants was nearly five times higher than in wild-type plants. These findings confirm the successful expression of the transferred gene and its role in enhancing drought tolerance. This result aligned with our expectations and confirmed the successful expression of the transferred gene. Other studies have documented similar results that have expressed this gene in different plants. Lin et al.67 reported that overexpressing SOD genes derived from Arabidopsis and winter squash improves chilling tolerance through ABA-sensitive transcriptional mechanisms in transgenic Arabidopsis. Findings indicated that Arabidopsis seedlings with elevated levels of AtSOD and CmSOD showed significantly higher tolerance to chilling stress and reduced oxidative damage compared with wild-type plants subjected to cold conditions. This evidence suggests that overexpressing AtSOD and CmSOD in Arabidopsis may facilitate chilling tolerance by effectively removing superoxide radicals (O2·−). Drought-induced ABA accumulation has been shown to activate antioxidant enzymes, including SOD, through ABA-dependent signaling cascades68. Thus, the upregulation of SmSOD in transgenic plants may function in coordination with ABA-mediated regulatory pathways to optimize stomatal behavior, sustain photosynthetic efficiency, and maintain redox homeostasis under drought conditions.

Gupta et al.69 showed that overexpressing pea-derived SOD in tobacco plants provided better protection against oxidative stress. The results revealed that SOD + plants maintained higher photosynthesis rates compared to SOD- plants at all growth stages, with SOD activity nearly three times higher in SOD + plants. Additionally, leaf discs from SOD- plants, which did not express pea SOD, exhibited significantly reduced photosynthesis. Various studies have emphasized the importance of SOD in conferring tolerance to abiotic stresses70,71. In this study, the highest enzymatic activity was observed in transgenic plants under drought conditions, 1.8 times higher than in wild-type plants.

Elevated SOD activity in the chloroplasts of transgenic tobacco plants provides better protection against membrane damage induced by methyl viologen (MV). Additionally, overexpression of Cu/Zn SOD improves plant resilience against photooxidative damage under intense light and low temperature conditions72. Transgenic plants co-expressing SOD and APX in chloroplasts exhibit greater tolerance to oxidative stress, with higher activities of both APX and SOD compared to wild-type plants73. Other studies have also reported increased SOD activity in transgenic plants under abiotic stress74,75.

Photosynthetic functionality and photoprotective mechanisms enhanced by SmSOD transformation in tobacco plants

The photosynthetic functionality of the plant was examined. The parameters investigated in this study included photosynthetic functionality (Fv/Fm), photoprotective mechanisms (NPQ), and stomatal characteristics (morphology, density, and dimensions). Regarding photosynthetic functionality, transgenic plants had higher values for Fv/Fm than wild-type plants. The higher Fv/Fm values observed in SmSOD-overexpressing tobacco plants indicate improved photosystem II (PSII) efficiency and reduced photoinhibition under drought stress. This enhancement suggests that SmSOD overexpression contributes to maintaining chloroplast integrity and protecting the PSII reaction centers from oxidative damage through efficient ROS scavenging. Van Beek et al.76 investigated the impact of overexpressing the SlNAC2 transcription factor on the photosynthetic apparatus of tobacco plants under water scarcity conditions. The results showed that both stomatal conductance and Fv/Fm were reduced in transgenic and wild-type plants under water stress, while these parameters remained stable in well-watered control plants. Jia et al.77 also demonstrated that Fv/Fm decreased under drought stress. In this study, Fv/Fm values declined in plants under stress treatments. Some studies reported a significant increase in Fv/Fm due to water scarcity78, while others found no significant changes79.

Regarding photoprotective mechanisms, higher NPQ levels were identified in transgenic plants exposed to water scarcity than in wild-type plants. NPQ refers to the fraction of light energy captured through antenna pigments that are not employed for electron transfer and dissipated as heat, mainly through carotenoids. Higher NPQ values are crucial for dissipating surplus energy80, which lets photosystems eliminate the extra energy input. The increased NPQ values in transgenic plants reflect a strengthened photoprotective mechanism, which facilitates the dissipation of excess excitation energy as heat, thereby preventing ROS overproduction. These physiological responses are closely linked to the antioxidant defense system, where SOD acts as the first enzymatic barrier converting superoxide radicals into less harmful molecules. Glowacka et al.81 showed that lowering excitation pressure in PSII through enhanced NPQ can effectively reduce excitation pressure under light conditions. In tobacco, NPQ enhancement was achieved by constitutively overexpressing PsbS in PSII82. Liu et al.83 explored the synergistic effects of cold and drought stress on photosynthetic activity and osmotic regulation in Elymus nutans Griseb, finding significant decreases in PSII electron transport rate and NPQ, indicating reduced energy dissipation and increased vulnerability of the photosystem. These results aligned with other studies showing no significant changes in Fv/Fm or NPQ under drought conditions84. Similar findings were reported in potatoes, chickpeas, tobacco, maize under drought, and soybeans exposed to cold stress85,86. Conversely, Gallie and Chen87 found that mutations in FSD2 led to elevated superoxide production, decreased chlorophyll content, and reduced PSII efficiency in Arabidopsis.

In examining stomatal characteristics, both transgenic and wild-type plants showed significantly lower stomatal density under water scarcity compared to control plants, while stomatal width was greater in stressed plants, with transgenic plants having wider stomata than wild-type plants. Water loss through stomatal transpiration is essential for regulating leaf temperature and indicates the plants’ ability to retain water and tolerate drought conditions88. Transgenic plants had higher stomatal conductance under drought stress compared to wild-type plants, and as stress intensified, significant changes were observed in stomatal length, width, and density.

Differing stomatal ROS responses influence stomatal closure in barley, revealing various drought regulation strategies89. Stomatal closure is one of the initial responses to water scarcity, significantly limiting photosynthesis90. Drought-tolerant genotypes exhibited slower wilting rates and increased water use efficiency (WUE), due to reduced water loss from stomatal closure, which helps maintain soil moisture. Under normal conditions, these genotypes showed higher stomatal width and greater density compared to drought-sensitive genotypes91.

Vulnerable wild barley genotypes showed higher stomatal density in response to drought stress, but a decrease in stomatal dimensions was observed92. In contrast, grapevine cultivars exhibited higher stomatal density and smaller stomata under drought conditions93. Zhang et al.94 showed that overexpression of TaMnSOD in transgenic cotton increased drought tolerance, leading to higher transpiration, stomatal conductance, and net photosynthesis. Similarly, Pal et al.95 demonstrated that overexpression of PaSOD in transgenic potatoes enhanced photosynthetic efficiency under drought stress. These results align with findings in tobacco, where successful transfer and expression of the SmSOD gene improved photosynthetic efficiency in transgenic plants.

Conclusion

Water scarcity and the increasing prevalence of drought have a profound impact on the environment, significantly affecting agriculture and food security. Addressing this issue is crucial to prevent further deterioration of global food supply systems. Biotechnological advancements, particularly in genetic engineering, offer promising approaches to mitigate these challenges. In this study, a key gene associated with tolerance to both biotic and abiotic stresses was transferred from a resistant plant species (Milk thistle) to a sensitive species (Tobacco). The SmSOD gene utilized in this research belongs to the Cu/ZnSOD type, which, compared to other SOD isoforms such as MnSOD and FeSOD, plays a broader role in regulating oxidative balance within various organelles, including chloroplasts, the cytosol, and peroxisomes. The use of the SmSOD gene as a Cu/ZnSOD represents a significant novelty in this study, as no prior research has investigated this specific isoform from Silybum marianum or evaluated its function in interspecies gene transfer. The objective of this research was to assess the effectiveness of this genetic modification in enhancing drought stress tolerance. The results demonstrated that SmSOD gene transfer positively influenced photosynthetic performance and photoprotective mechanisms in tobacco plants, thereby improving their stress resilience. Based on these findings, applying this gene transfer strategy to other sensitive crop species is recommended. Incorporating stress-tolerant genes from resistant plants could potentially enhance crop resilience to environmental stresses, thereby promoting sustainable agriculture and contributing to global food security.