Heterologous Expression of Sorghum bicolor PIP1-3 Gene Improves Drought Tolerance in Arabidopsis and Rapeseed

Abstract

Aquaporins are key membrane proteins that mediate water transport in plants and are indispensable for maintaining cellular water homeostasis and normal physiological processes. This study investigated the function of SbPIP1-3, an aquaporin gene isolated from drought-tolerant Sorghum bicolor. Bioinformatics analysis, subcellular localization, and heterologous expression of SbPIP1-3 were performed in Saccharomyces cerevisiae, Arabidopsis thaliana, and rapeseed. Sequence analysis revealed that SbPIP1-3 encodes a basic hydrophobic protein targeted to the plasma membrane, a finding further corroborated by subcellular localization assays. In yeast expression assays, SbPIP1-3-transformed strains retained viability under osmotic stress induced by 1.2 M mannitol, whereas non-transgenic control strains failed to survive. In Arabidopsis and rapeseed experiments, the SbPIP1-3 overexpression enhanced drought tolerance (improved germination, root growth, antioxidant enzyme activity, proline content, PSII repair capacity, and survival after drought–rewatering) and reduced intracellular H₂O₂ accumulation. Transcriptome profiling of drought-stressed transgenic Arabidopsis and control plants demonstrated significant upregulation of mostly stress-responsive pathways (e.g., MAPK signaling pathway and hormone signaling pathways) and key drought-tolerance genes (e.g., SNRK2-2, SOD1, APX3, GPX3, P5CS1). Collectively, these findings suggest that SbPIP1-3 enhances plant drought tolerance through the following mechanisms: improving transmembrane water transport efficiency to sustain cellular osmotic balance; activating the antioxidant defense system to increase enzyme activity and mitigate reactive oxygen species (ROS) accumulation; optimizing photosynthetic protection mechanisms to preserve the structural and functional integrity of PSII; and regulating the expression of stress-responsive signaling pathways and associated functional genes.

1. Introduction

Drought is a widely distributed abiotic stress globally, exerting significant adverse effects on the growth and development of crops and directly or indirectly leading to reduced crop yield and quality [1]. Under drought stress, the contents of malondialdehyde (MDA) and reactive oxygen species (ROS) increase in plants, triggering membrane lipid peroxidation and cellular damage, which results in decreased chlorophyll content, reduced photosynthetic rate, and an induced imbalance of endogenous hormone homeostasis. In response, plants can enhance their osmotic adjustment capacity by accumulating osmotic adjustment substances, such as soluble proteins and proline (Pro), and increasing the activities of various antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), to scavenge excess ROS [2,3,4,5]. In rapeseed, these physiological and biochemical regulatory processes help to improve water-use efficiency and alleviate damage caused by drought stress.

Aquaporins (AQPs) are members of the major intrinsic protein (MIP) superfamily localized on the plasma membrane or tonoplast. Their core function is to mediate the efficient transmembrane transport of water and small molecules, playing a crucial role in regulating plant water balance, nutrient uptake, and stress responses [6]. Based on their sequence homology and subcellular localization characteristics, plant AQPs can be classified into multiple subfamilies, including plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs). Among these, the PIP subfamily exhibits the closest association with plant abiotic stress tolerance and can be further subdivided into two subfamilies: PIP1 and PIP2 [7,8].

The functional mechanisms of PIP2 subfamily members in crop stress resistance have been extensively elucidated. Studies have shown that PIP2s participate in the regulation of stress responses through interactions with key regulatory factors and mediation of water and signaling molecule transport. For example, in a genome-wide association study in sorghum, Zhang et al. found that the major quantitative trait locus for alkaline tolerance, AT1, can interact with PIP2s on the plasma membrane to alleviate alkali stress-induced cellular oxidative damage by promoting H₂O₂ efflux. Introduction of the sorghum AT1 gene into crops such as rice and wheat significantly improved their yields in saline–alkali soils [9]. Furthermore, overexpression and functional verification studies of PIP2 genes from various species, including rice, apple, wheat, soybean, switchgrass, and jatropha, have demonstrated that overexpressing plants can significantly enhance their tolerance to drought or salt stress through pathways such as improving water transport efficiency and increasing antioxidant enzyme activity [10,11,12,13,14,15,16].

Functional research on the PIP1 subfamily is relatively lagging that of the PIP2 subfamily. Members of the PIP1 subfamily possess the typical structural characteristics of the MIP superfamily. They exert numerous regulatory roles in mediating plant salt and drought tolerance, with functional mechanisms at multiple levels, such as maintenance of water homeostasis, regulation of signal transduction, and activation of antioxidant systems [7,8,17,18,19]. Functional characterization of PIP1 family members and transformation verification of heterologous PIP1 genes have confirmed their stress resistance potential: AtPIP1-4 from Arabidopsis can interact with the bacterial protein Hpa1 to improve the transport efficiency of CO₂ and water across the cell membrane and enhance the photosynthetic system’s stability to support energy metabolism under stress conditions [20]; overexpression of heterologous PIP1 genes from maize, wheat, broad bean, and Thellungiella halophila in Arabidopsis significantly enhanced the salt and drought tolerance of transgenic plants, manifested as phenotypic advantages such as increased survival rate and alleviated growth inhibition [21,22,23,24].

At the level of stress-resistant molecular mechanisms, PIP1 genes coordinate to regulate plant stress responses through multiple pathways: the first pathway is through involvement in the regulation of stomatal movement. PIP1 genes not only regulate water transport between leaf veins and guard cells but also increase the permeability of mesophyll cell and guard cell membranes to CO₂, thereby improving the catalytic efficiency of the Rubisco enzyme and potentially adapting to environmental changes by regulating plant transpiration efficiency [25]. The second pathway is through enhancement of the antioxidant defense system. In Arabidopsis overexpressing ZmPIP1-1, the activities of antioxidant enzymes such as CAT and SOD are significantly increased under salt and drought stress, which helps to effectively scavenge excess ROS and reduce membrane damage caused by MDA accumulation [21,26]. The third pathway is significant cross-species functional conservation: heterologous expression of PIP1 genes from wheat, maize, broad bean, and Thellungiella halophila in Arabidopsis can significantly improve the stress resistance of transgenic plants, confirming that the function of PIP1 genes is highly conserved between monocotyledonous and dicotyledonous plants [21,22,23,24]. The aforementioned studies indicate that plant PIP1 genes constitute the core nodes in the plant stress-resistant regulatory network through a multi-dimensional regulatory mechanism of “water transport–signal transduction–metabolic regulation.” Their functional characterization provides important genetic resources and theoretical basis for the genetic improvement of crop stress resistance.

Focusing on the SbPIP1-3 gene, the present study first validated its basic stress resistance function using the Saccharomyces cerevisiae heterologous expression system. Furthermore, SbPIP1-3 was transformed into Arabidopsis and rapeseed, and its regulatory role in drought tolerance across different crops was systematically verified at multiple levels, including seed germination, seedling growth, physiological indices, and photosynthetic characteristics. Additionally, combined with transcriptome sequencing analysis, the molecular pathways and key differentially expressed genes (DEGs) underlying SbPIP1-3-mediated plant drought tolerance were elucidated. The present study aims to clarify the drought tolerance function and regulatory mechanism of SbPIP1-3, enrich the functional understanding of the plant PIP1 subfamily, and provide important genetic resources and theoretical support for the molecular breeding of drought-tolerant crops.

2. Results

2.1. Characterization of SbPIP1-3 Protein

Amino acid sequence analysis revealed that the S. bicolor aquaporin gene SbPIP1-3 encodes a polypeptide consisting of 288 amino acids, with a molecular weight of approximately 30 kDa and an isoelectric point (pI) of 8.99, classifying it as a basic protein. The grand average of hydropathy (GRAVY) value of this protein is 0.376, and the lipid solubility index is 95.90, suggesting strong hydrophobic characteristics. The instability index is 28.78 (<40), categorizing it as a stable protein. Hydrophobicity analysis using the ProtScale software showed distinct hydrophobic regions (positive values) and hydrophilic regions (negative values) in the protein sequence, consistent with the characteristics of a typical membrane protein: the hydrophobic segments are usually embedded within the membrane, while the hydrophilic regions are exposed on the membrane surface (Figure 1A). In conclusion, SbPIP1-3 is identified as a stable, basic, hydrophobic membrane protein, and its structural features are consistent with those of the aquaporin family.

The multiple sequence alignment results (Figure 1B) show that the SbPIP1-3 protein contains the characteristic domains GGGANXXXXGY and TGI/TNPARSL/FGAAI/VI/VF/YN, indicating that it belongs to the plasma membrane aquaporin family. These signature sequences serve as important criteria for identifying plasma membrane aquaporin genes and their classification, and they are also key sequence units responsible for the function and regulation of plasma membrane aquaporins. Two inverted NPA (Asn-Pro-Ala) conserved domains, which form the hydrophilic channel of this aquaporin. Additionally, on the periplasmic side near the NPA motifs, there is an aromatic/arginine (Ar/R) selectivity filter region, this region acts as a selectivity filter for substrates. Conserved domain analysis of the SbPIP1-3 protein using the NCBI Conserved Domains Database revealed that it contains a typical signature sequence of the major intrinsic protein (MIP) superfamily, indicating that it is a member of the MIP superfamily.

Signal peptide prediction using the SignalP 4.1 software indicated that SbPIP1-3 lacks a signal peptide, suggesting it is a non-secretory protein. Transmembrane domain analysis of SbPIP1-3 with TMHMM software (version 2.0) revealed a high amino acid count of 129.30 in membrane helices, with six potential transmembrane helices (TMs). The amino acid segments in regions such as 1–54, 115–134, and 197–288 are located inside the cell membrane, while the segments in regions like 78–91, 153–178, and 232–256 are located outside the cell membrane. Overall, the protein forms a specific distribution pattern across the cell membrane through six transmembrane helices, with the N-terminus more inclined to be positioned inside the cell membrane.

2.2. Expression of SbPIP1-3 Gene Enhances Drought Tolerance in Yeast

On mannitol-free synthetic galactose uracil-deficient (SG/-U) medium, yeast harboring the recombinant plasmid pYES2-SbPIP1-3 showed no significant difference in colony morphology compared with yeast carrying the empty pYES2 vector (Figure 2). However, when the medium was supplemented with a final mannitol concentration of 0.8, 1.0, or 1.2 mol/L, and the initial culture density was adjusted to OD600 = 0.0002, the colony count of pYES2-SbPIP1-3 transformants was significantly higher than that of the empty vector control (pYES2). This result indicates that heterologous expression of the SbPIP1-3 gene significantly enhances osmotic stress-induced drought tolerance in S. cerevisiae.

2.3. Subcellular Localization of SbPIP1-3

The subcellular localization of the SbPIP1-3 protein is shown in Figure 3. In the empty vector control group (Figure 3A), the GFP signal was distributed in both the nucleus and the plasma membrane, while the GFP signal in the experimental group (Figure 3B) was specifically localized to the plasma membrane. The RFP signal was stably localized to the plasma membrane in both groups, forming clear cell boundaries and verifying the effectiveness of the membrane labeling system. In the control group (Figure 3A), there was no significant colocalization of GFP and RFP signals (only random, non-specific overlap was observed in the Merge panel), indicating that the empty vector-derived GFP itself was not localized to the plasma membrane. In the experimental group (Figure 3B), the two signals showed complete colocalization, displaying uniform membranous composite fluorescence (yellow-green and red overlap). This result demonstrates that the SbPIP1-3 protein is specifically localized to the plasma membrane.

Figure 2. Drought treatment of SbPIP1-3-transformed yeast (INVSc1).

Figure 2. Drought treatment of SbPIP1-3-transformed yeast (INVSc1).

Figure 3. Subcellular localization of SbPIP1-3 protein. GFP: green fluorescent protein; RFP: red fluorescent protein; Bright: Brightfield; Merge: merged image. Panels (A,B) represent the subcellular localization of the pCAMBIA1302 empty vector and the pCAMBIA1302-PIP1-3 fusion protein, respectively. Scale bar = 20 μm.

Figure 3. Subcellular localization of SbPIP1-3 protein. GFP: green fluorescent protein; RFP: red fluorescent protein; Bright: Brightfield; Merge: merged image. Panels (A,B) represent the subcellular localization of the pCAMBIA1302 empty vector and the pCAMBIA1302-PIP1-3 fusion protein, respectively. Scale bar = 20 μm.

2.4. Expression of SbPIP1-3 Enhances Germination Rate and Drought Tolerance in Arabidopsis Under Drought Stress

Arabidopsis seeds were germinated in media supplemented with different concentrations of PEG 6000 to simulate drought stress. At 0% and 10% PEG 6000, no difference in germination rate was observed between the non-transgenic control and the transgenic lines OE1 and OE2, with all lines reaching nearly 100% germination (Figure 4A,B,E,F). However, at 15% PEG 6000, the final germination rate of OE1 and OE2 remained at 100%, compared to 76% for the control (Figure 4C,G). Under more severe stress with 20% PEG 6000, the germination rates of OE1 and OE2 were 98% and 100%, respectively, while that of the control dropped to 56% (Figure 4D,H).

Under PEG 6000-induced drought stress, root growth was also affected. In the absence of PEG 6000, no significant differences in root length were detected between the control and transgenic lines (Figure 5A,D). However, at 15% and 20% PEG 6000, the root length of the WT was significantly shorter than that of OE1 and OE2 (Figure 5B–D). To further investigate the physiological basis of this enhanced stress tolerance, we measured the activities of POD and CAT and the contents of proline and H₂O₂ in Arabidopsis plants grown under 20% PEG 6000. The measurement showed that POD and CAT activities, as well as Pro content, were significantly higher in OE1 and OE2 than in the WT, while H₂O₂ accumulation was markedly lower (Figure 5E–H).

To comprehensively assess drought tolerance, the transgenic lines OE1 and OE2 and WT controls were subjected to artificial drought stress by withholding water. By the seventh day of drought treatment, the WT control plants exhibited more severe wilting symptoms than OE1 and OE2 plants (Figure 6B), and this difference became more pronounced by the ninth day (Figure 6C). Consistent with these phenotypic observations, the control plants accumulated significantly higher levels of H₂O₂ and MDA and exhibited lower SOD activity compared to the transgenic lines (Figure 6F–H). These results suggest that SbPIP1-3 overexpression enhances SOD activity, improves reactive oxygen species scavenging capacity, and reduces oxidative damage under drought stress. One day after rewatering, 76.67% of OE1 plants (92 out of 120) and 92.62% of OE2 plants (113 out of 122) fully recovered, whereas only 10.66% of non-transgenic plants (13 out of 125) resumed normal growth (Figure 6E).

Collectively, these results demonstrate that heterologous expression of the SbPIP1-3 gene significantly enhances drought tolerance in Arabidopsis.

2.5. Photosynthetic Characteristics of Transgenic Arabidopsis Under Drought Stress

The QY_max (Maximum Quantum Yield) reflects the efficiency of photochemical energy conversion, with higher values indicating greater efficiency. Under well-watered conditions, the WT and two overexpression Arabidopsis lines (OE1 and OE2) all showed QY_max values above 0.80 (Figure 7(C2)). After 7 days of water withholding, the QY_max decreased across all genotypes; however, the decline was less pronounced in the transgenic lines OE1 and OE2 compared to the WT. The mean QY_max values of OE1 and OE2 (0.743 and 0.675, respectively) were significantly higher than that of the WT (0.437), indicating that the photosystem II (PSII) core proteins in the transgenic plants maintain better structural stability under drought stress.

The Y_NPQ (Yield of Non-photochemical Quenching at Steady State) represents the proportion of absorbed light energy dissipated as heat via regulatory mechanisms, serving as a key process for protecting PSII. Under normal irrigation, the WT, OE1, and OE2 lines showed Y_NPQ values between 0.302 and 0.311 (Figure 7(D2)). After 7 days without watering, the Y_NPQ declined in all lines, though the transgenic lines OE1 and OE2 exhibited a smaller reduction; their mean Y_NPQ values (0.353 and 0.295) were significantly higher than that of the WT (0.06).

The qN_Lss (Non-photochemical Quenching Coefficient at Steady State) reflects the capacity for non-photochemical quenching; higher values indicate more efficient dissipation of excess light energy. Under well-watered conditions, the WT, OE1, and OE2 lines all displayed qN_Lss values above 0.58 (Figure 7(E2)). Drought stress led to a decrease in qN_Lss across all genotypes, but the transgenic lines OE1 and OE2 experienced a smaller decline; their average qN_Lss values (0.525 and 0.503) were significantly higher than that of the WT (0.416).

The RFd (Relative Fluorescence Decline at Steady State) is an indicator of the overall capacity of the dark reactions (carbon assimilation) to drive photochemical processes; higher values suggest stronger photosynthetic performance. Under normal watering conditions, the WT, OE1, and OE2 lines all had RFd values exceeding 0.22 (Figure 7(F2)). After 7 days of drought treatment, RFd decreased in all lines, but the transgenic plants OE1 and OE2 showed a more moderate reduction; their mean RFd values (0.202 and 0.178) were significantly higher than that of the WT (0.07).

In summary, under drought stress, transgenic Arabidopsis plants exhibit enhanced photoprotection mechanisms, improved non-photochemical quenching capacity, and reduced generation of reactive oxygen species (e.g., H₂O₂). These improvements collectively contribute to the elevated overall photosynthetic performance and increased drought tolerance in transgenic Arabidopsis plants.

2.6. Water Loss Rate and Stomatal Aperture in Arabidopsis Detached Leaves

Assessment of the water loss rate in detached leaves (Figure 8C) revealed that although all plants showed a progressive increase in water loss over time, the transgenic lines exhibited significantly lower water loss rates compared to the WT plants. Specifically, the water loss rate of OE1 was 30.11%, 29.90%, and 21.24% lower than that of the WT control at 3 h, 4 h, and 5 h, respectively. Similarly, OE2 showed significant reductions of 13.25%, 15.08%, and 11.96% at the corresponding time points.

Leaf water loss is largely regulated by stomatal aperture, a process influenced by Abscisic Acid (ABA) signaling [27]. When epidermal cells were treated with water, no significant differences in stomatal aperture (width/length) were detected among the WT, OE1, and OE2 lines, with values ranging from 33.05 to 35.33. However, following treatment with 10 μmol·L⁻¹ ABA, stomatal aperture was markedly reduced (Figure 8A,B). The apertures in OE1 and OE2 measured 17.28 and 14.31, respectively, with both values being significantly smaller than that of the WT control (26.74) (Figure 8B,D). These results suggest that ABA treatment induces a greater reduction in stomatal aperture in the transgenic plants than in the WT. This enhanced stomatal closure likely contributes to reduced water loss, thereby improving the transgenic lines’ drought tolerance capacity.

2.7. Heterologous Expression of SbPIP1-3 Improves Drought Tolerance in Rapeseed

Seeds of the non-transgenic WT and two transgenic SbPIP1-3 rapeseed lines (PIP#3 and PIP#9) were germinated in boxes containing 0%, 10%, 15%, or 20% PEG 6000. At 0% and 10% PEG 6000, the final germination rates of the WT, PIP#3, and PIP#9 all reached 100%. When PEG 6000 was increased to 15%, both PIP#3 and PIP#9 attained 100% germination by day 7, whereas the final germination rate of the WT was only 68%. At 20% PEG 6000, the final germination rates of PIP#3 and PIP#9 were 95% and 100%, respectively, but that of the WT dropped markedly to 60%. These results demonstrate that SbPIP1-3 significantly enhances rapeseed seed germination under drought stress (Figure 9).

Drought stress was simulated in rapeseed seedlings using different concentrations of PEG 6000 solution. Under 0% and 15% PEG 6000 conditions, no significant differences were observed between PIP#3, PIP#9, and WT (Figure 10A–D). When the PEG 6000 concentration was increased to 20% and 25%, the WT plants showed wilting, whereas the PIP#3 and PIP#9 plants maintained normal growth (Figure 10E–H).

After three days of treatment with 20% PEG 6000, leaves were collected to measure SOD enzyme activity, as well as MDA, H₂O₂, and SSC levels. The measurement showed that SOD activity was significantly higher in the transgenic lines PIP#3 and PIP#9 than in the WT (Figure 11A,D), while the MDA and H₂O₂ contents were significantly lower than those in the WT (Figure 11B,C). These findings suggest that the transgenic plants enhance drought tolerance by increasing antioxidant enzyme activity and reducing membrane lipid peroxidation and reactive oxygen species accumulation.

The development of an extensive root system is critical for drought resistance in rapeseed plants. Under control conditions (0% PEG 6000; Figure 12A), the root morphology and length of the WT plants did not differ significantly from those of the PIP#3 and PIP#9 transgenic lines. In contrast, under higher PEG 6000 concentrations (15%, 20%, and 25%; Figure 12B–D), the transgenic lines displayed superior root growth, as evidenced by their significantly enhanced root length, fresh weight, and dry weight compared to the WT control (Figure 12E–G). These results suggest that the improved drought tolerance of the transgenic lines is due to the expression of the exogenous SbPIP1-3 gene, which promotes root development and consequently boosts water uptake efficiency under drought stress.

To further evaluate the drought tolerance of the transgenic rapeseed lines, plants were subjected to a drought stress regime by withholding water. After 9 and 11 days of water withdrawal, all plants showed signs of wilting due to water deficit; however, wilting was more severe in the WT plants than in the transgenic lines (Figure 13B,C). Watering was resumed on day 12. By the third day of rewatering, all 67 PIP#9 plants and 67 of 68 PIP#3 plants had fully recovered; in contrast, only 15 of 68 WT plants had fully recovered (Figure 13F).

On day 9 of drought stress, the leaf H₂O₂ content and SOD activity were measured. The WT plants accumulated significantly higher levels of H₂O₂ (Figure 13G) and exhibited significantly lower SOD activity compared to the PIP#9 and PIP#3 lines (Figure 13H). These results suggest that the expression of the transgene enhances drought tolerance by elevating SOD activity, which in turn reduces reactive oxygen species and mitigates oxidative cellular damage.

2.8. Photosynthetic Characteristics of Transgenic Rapeseed Plants Under Drought Stress

Under well-watered conditions, the control WT and rapeseed transgenic lines (PIP#3, PIP#9) showed average QY_max values above 0.800, Y_NPQ values above 0.331, qN_Lss values above 0.440, and RFd values above 0.238 (Figure 14(C1–F1)). After 7 days of water withdrawal, all the above chlorophyll fluorescence parameters decreased across the genotypes, but the reductions in the transgenic lines PIP#3 and PIP#9 were less pronounced than those in the WT. Specifically, the average QY_max values of PIP#3 and PIP#9 were 0.746 and 0.767, respectively, which were significantly higher than that of the WT (0.633) (Figure 14(C2)). The Y_NPQ averages were 0.276 and 0.288, significantly higher than the value of 0.206 for the WT (Figure 14(D2)). The average qN_Lss values were 0.376 and 0.401, significantly exceeding the WT’s value of 0.298 (Figure 14(E2)). The average RFd values were 0.216 and 0.209, also significantly higher than the value of 0.113 observed in the WT (Figure 14(F2)). These results indicate that under drought stress, the transgenic plants exhibit enhanced photoprotective mechanisms, improved light-use efficiency, and consequently, better drought tolerance.

2.9. Differentially Expressed Genes in SbPIP1-3 Transgenic Arabidopsis Under Drought Stress

To further understand the drought tolerance mechanism mediated by the SbPIP1-3 gene in transgenic plants, we conducted a genome-wide expression study using RNA-seq technology on SbPIP1-3 transgenic Arabidopsis plants and the WT control. The high-quality sequencing reads obtained after quality control were aligned to the reference genome, and PCA confirmed the high integrity and accuracy of the transcriptome sequencing results. To investigate the impact of heterologous SbPIP1-3 gene expression in Arabidopsis, Gene Ontology (GO) analysis was performed to examine differences between the WT and transgenic plants. The GO analysis revealed that the differentially expressed genes (DEGs) were primarily clustered into three major categories: Molecular Function (MF), Cellular Component (CC), and Biological Process (BP). The main biological processes identified were “response to stimulus,” “cellular biosynthetic process,” and “response to stress.” In the Molecular Function category, DEGs were associated with “catalytic activity” and “ion binding.” Finally, the core cellular components were localized to the “membrane system” (Figure 15A–C).

KEGG enrichment analysis of the DEGs identified three significantly enriched pathways: plant hormone signal transduction, MAPK signaling pathway–plant, and plant–pathogen interactions (Figure 15D–F). To screen candidate DEGs under drought stress, we analyzed the expression profiles of protein-coding genes based on the FPKM values and selected the top five significantly enriched DEGs (p < 0.05) from the KEGG pathways. The analysis showed that the expression of several genes was significantly upregulated in the SbPIP1-3 transgenic plants compared to the non-transgenic controls, including SNRK2-2, SNRK2-10, and SNRK2-3 from the sucrose non-fermenting 1-related protein kinase 2 family; P5CS1 and P5CS2 from the delta-1-pyrroline-5-carboxylate synthase family; genes from the Betaine Aldehyde Dehydrogenase (BADH) family; and antioxidant enzyme genes such as SOD1, POD3, and CAT2 (Figure 16).

3. Discussion

In recent years, the role of AQPs in plant responses to abiotic stress has attracted increasing attention. Studies have shown that AQPs play a central regulatory role in physiological processes such as transmembrane water transport, cellular water balance, and responses to abiotic stress [28]. Among them, the PIPs subfamily is particularly closely associated with plant abiotic stress responses [29]. Multiple studies have reported that overexpression of PIP genes, such as OsPIP1, OsPIP2, VfPIP1, and BnPIP1, in plants, including rice, legumes, and rapeseed, can significantly enhance tolerance to osmotic or drought stress [23]. Furthermore, heterologous expression of pumpkin CdPIP2-1 or wild soybean GsPIP2-7 in Arabidopsis, as well as overexpression of rapeseed BnPIP1 in tobacco, effectively improved the drought tolerance of transgenic plants [30,31]. In this study, using the drought-tolerant crop sorghum as the gene source, we systematically validated the drought resistance function of the SbPIP1-3 gene in yeast, Arabidopsis, and rapeseed. This work expands the current understanding of the functional conservation of the PIP gene family across different species and provides new insights into the molecular mechanisms of plant adaptation to drought.

The stability of the photosynthetic system serves as an important basis for evaluating plant drought resistance. Parameters such as the QY_max, Y_NPQ, qN_Lss, and RFd directly reflect the response capacity of the photosynthetic apparatus under stress conditions [32,33,34]. This study demonstrated that, under drought stress, both SbPIP1-3 transgenic Arabidopsis and rapeseed exhibited significantly superior performance in these four chlorophyll fluorescence parameters compared to the non-transgenic controls. These results suggest that SbPIP1-3 may help to maintain the structural integrity of the PSII reaction center, enhance the efficiency of light energy dissipation, and reduce ROS-induced damage to the photosynthetic system, thereby ensuring basic photosynthetic function under drought conditions and ultimately improving plant drought tolerance.

SnRK2 represents a class of serine/threonine protein kinases involved in ABA signaling and osmotic stress responses, playing a broad role in plant adaptation to stresses such as salinity and drought [35]. P5CS is a key rate-limiting enzyme in proline biosynthesis, catalyzing the conversion of glutamate to Δ¹-pyrroline-5-carboxylate, and its high expression significantly promotes proline accumulation [36]. As a highly soluble, neutral osmoprotectant, proline effectively lowers cellular osmotic potential, which helps to maintain turgor pressure under drought conditions, thereby supporting essential physiological processes such as cell elongation, stomatal opening, and photosynthesis. In addition, proline can act as a molecular chaperone, stabilizing protein structures via hydrogen bonding and facilitating their proper refolding after rehydration. It also interacts with membrane lipids to maintain membrane system integrity and alleviate drought-induced increases in membrane permeability [37]. The BADH gene is involved in the synthesis of glycine betaine, an important osmoprotectant and macromolecule stabilizer that, under drought conditions, effectively protects thylakoid membrane structure and the activity of key enzymes such as Rubisco. This study showed that heterologous expression of SbPIP1-3 significantly upregulates the expression of SnRK2, P5CS, and BADH family genes, thereby promoting the accumulation of proline and glycine betaine and, consequently, enhancing plant drought tolerance through multiple mechanisms, including osmotic adjustment, antioxidant defense, and macromolecule protection.

Under drought stress, ROS are abundantly generated in organelles such as chloroplasts and mitochondria. The efficient expression of SOD genes enables timely scavenging of superoxide anions, thereby preventing the formation of more destructive hydroxyl radicals and protecting biomacromolecules and membrane structures. The H₂O₂ produced by the SOD-catalyzed reaction is subsequently eliminated by enzymes such as POD and CAT. This study revealed that the expression of POD and CAT genes was significantly upregulated in transgenic plants, indicating an enhanced capacity to remove H₂O₂ and repair membrane lipid peroxidation damage. This ability is crucial for maintaining the stability of cell membrane under dehydration conditions. We propose that SbPIP1-3 may systematically activate the antioxidant defense system, enabling transgenic plants to scavenge various forms of ROS more rapidly and efficiently, thereby markedly reducing oxidative damage and improving these plants’ survival capability under drought stress.

Under the drought stress conditions applied in this study, SbPIP1-3 transgenic Arabidopsis and rapeseed plants demonstrated distinct physiological superiority. When subjected to drought simulated by 20% PEG 6000, the transgenic lines developed significantly longer roots, exhibited higher activities of POD and CAT, accumulated more proline, and showed reduced H₂O₂ levels compared to the wild-type plants. Following nine days of natural drought treatment, the wild-type plants displayed more pronounced wilting than the transgenic lines, along with a significantly lower survival rate after rewatering. Concurrently, the leaf MDA content was markedly lower in the transgenic plants than in the wild-type controls. Collectively, these results indicate that SbPIP1-3 enhances overall drought tolerance by strengthening osmotic adjustment and antioxidant capacity, thereby reducing ROS-mediated membrane damage and improving the plant’s ability to withstand water deficit.

4. Materials and Methods

4.1. Plant Materials and Microbial Strains

The SbPIP1-3 gene (GenBank Accession No. XM_002446884.2) from sorghum was chemically synthesized. The primers p-PIP-F and p-PIP-R (Table S1) were designed to introduce Xba I and BamH I restriction sites at the two ends of the SbPIP1-3 gene via PCR. The sequence with Xba I and BamH I sites was subsequently cloned into the pYES2 and pCAMBIA1302 vectors, resulting in the recombinant plasmids pYES2-PIP1-3 and pCAMBIA1302-PIP1-3, respectively. The pYES2-PIP1-3 plasmid was transfected into the S. cerevisiae strain INVSc1 using the lithium acetate method [38]. Meanwhile, the pCAMBIA1302-PIP1-3 plasmid was introduced into the Arabidopsis ecotype Col-0 and B. napus cultivar Xiangyou 18 via Agrobacterium-mediated transformation. SbPIP1-3 positive clones were identified by PCR with the primers PIP-F and PIP-R (Table S2). The following transgenic materials were used to investigate SbPIP1-3-mediated drought tolerance: the transformed INVSc1 yeast strain, two independent B. napus transgenic lines (PIP#3 and PIP#9), and two Arabidopsis transgenic lines (OE1 and OE2).

4.2. Bioinformatics Analysis

The conserved domains of the protein were identified using the online Conserved Domains Database (CDD). The isoelectric point (pI) and relative molecular weight were predicted with the ExPASy ProtParam tool. The transmembrane domains were analyzed via the TMHMM Server v. 2.0, while the hydrophilicity/hydrophobicity profile of the amino acid sequence was assessed using ProtScale. Characteristic sequence motifs and structural features were predicted using the Prosite database.

4.3. Yeast Drought Stress Assay

The yeast strain INVSc1 was transformed with either the pYES2-PIP1-3 plasmid or the empty pYES2 vector as a negative control. The transformants were serially diluted to OD600 of 0.2, 0.02, 0.002, and 0.0002. The aliquots (7.5 μL) of each dilution were spotted onto SG/-Ura solid medium plates supplemented with 0, 0.8, 1.0, or 1.2 M mannitol. After 3-day incubation at 28 °C, the growth phenotypes were recorded.

4.4. Subcellular Localization

The pCAMBIA1302-PIP1-3 plasmid and the pRFP vector (a plasma membrane marker with mCherry tag) were individually transfected into Agrobacterium tumefaciens GV3103. These bacterial suspensions were co-infiltrated into Nicotiana benthamiana leaves. After two days, confocal laser scanning microscopy was used to visualize the co-expressed GFP and RFP signals to determine PIP1-3 localization.

4.5. Seed Germination Assay Under Drought Stress in Transgenic SbPIP1-3 Plants

Seeds of transgenic and WT Arabidopsis (control) and rapeseed were surface-sterilized with 75% ethanol. The sterilized Arabidopsis seeds were sown on 1/2 MS solid medium supplemented with 0%, 10%, 15%, or 20% PEG 6000, whereas rapeseed seeds were placed in germination boxes containing the same concentrations of PEG 6000. All plates and boxes were incubated in a growth chamber under a 12-h light/12-h dark cycle with a light intensity of 200 μE m⁻² s⁻¹. Germination was recorded every 24 h for 7 days. The final germination rate was calculated, with each treatment consisting of three biological replicates.

4.6. Drought Treatment of SbPIP1-3 Transgenic Plants

Seeds of WT (control) and SbPIP1-3 transgenic Arabidopsis were sterilized and sown on 1/2 MS solid medium. After 5 days of growth, uniformly developed seedlings were transferred to 1/2 MS medium containing 0%, 15%, or 20% PEG 6000 to simulate drought stress. For rapeseed, transgenic SbPIP1-3 and non-transgenic control seeds were sown in seedling trays. At the three-leaf stage, the plants were irrigated daily with 10 mL of PEG 6000 solution at a concentration of 0%, 10%, 15%, 20%, or 25% for 10 days to induce drought stress. In a parallel pot experiment, both transgenic Arabidopsis and rapeseed plants were grown in a 2:1 mixture of nutrient soil and vermiculite under controlled conditions (as described in Method Section 4.5). After 30 days of growth, watering was withheld for 10 days, followed by rewatering. Plant growth performance during drought and after recovery was monitored and recorded. In all assays, drought tolerance was comprehensively evaluated based on phenotypic observations and plant growth status.

4.7. Analysis of Water Loss Rate and Stomatal Aperture

The water loss rate of detached leaves was measured following the method of Xiao et al. [39]. Rosette leaves of similar size were collected from both SbPIP1-3 transgenic and WT Arabidopsis, and their initial fresh weight was recorded. The leaves were placed adaxial side up under controlled conditions (25 °C, 50% relative humidity) and weighed at 0.5, 1, 2, 3, 4, and 5 h. The experiment included three biological replicates. The water loss rate was calculated as follows: (initial weight − weight at time point) / initial weight.

Stomatal aperture was assessed according to Wang et al.’s method [40]. Rosette leaves from SbPIP1-3 transgenic and WT Arabidopsis were floated in a solution containing 10 mmol L⁻¹ KCl, 50 mmol L⁻¹ CaCl₂, and 10 mmol L⁻¹ MES (pH 6.15). The leaves were first incubated in an illuminated growth chamber at 22 °C under 100 μmol m⁻² s⁻¹ light for 3 h. Then, 10 μmol L⁻¹ ABA was added, and incubation continued for an additional 3 h under the same conditions. Leaves not subjected to ABA treatment served as the control. The lower epidermis was carefully peeled and examined using an inverted fluorescence microscope (Murzider, Shenzhen, China). Three fields of view were analyzed per group, with ten stomata randomly measured per field. The stomatal length and width were determined using the IMAGEJ software (version. 2.15.0), and the stomatal aperture is expressed as the width-to-length ratio.

4.8. Determination of Physiological Parameters

Following drought treatment, leaf samples were collected from both SbPIP1-3 transgenic and WT Arabidopsis and rapeseed plants. Healthy, intact, and fully expanded functional leaves were selected; namely, the 3rd–4th leaves for Arabidopsis and the 4th–5th leaves for rapeseed. During sampling, the midrib was avoided, and mesophyll tissue from the middle part of the leaves was excised. The leaf samples were rapidly rinsed with sterile water to remove surface impurities, blotted dry with absorbent paper, immediately aliquoted into pre-chilled cryovials, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.

Relevant physiological indices were determined using commercial kits purchased from Grace Biotechnology Co., Ltd. (Suzhou, China), following the manufacturer’s instructions. POD activity was measured via the guaiacol method; CAT activity via the ultraviolet spectrophotometric method; SOD activity via the nitroblue tetrazolium (NBT) photoreduction method; MDA content via the thiobarbituric acid (TBA) colorimetric method; H₂O₂ content via the titanium salt colorimetric method; Pro content via the acid ninhydrin colorimetric method; and soluble sugar content (SSC) via the anthrone colorimetric method.

Photosynthetic parameters were measured using a FluorCam 1300 fluorescence imaging system (PSI, Prague, Czech Republic). Prior to measurement, the plants were dark-adapted in a dark room for 30 min. Whole plants were selected to determine the QY_max, Y_NPQ, Rfd, and qN_Lss, which were used to evaluate leaf photosynthetic efficiency among different treatments. All assays included three biological replicates, with at least three plants per biological replicate.

4.9. Transcriptome Analysis

Transcriptome analysis was conducted to identify differentially expressed genes (DEGs) between SbPIP1-3 transgenic and WT Arabidopsis plants. The plants were subjected to two treatments: 7-day drought stress simulated by 20% PEG 6000 and regular watering (control). The experiment included three biological replicates, with leaf samples collected from three plants per biological replicate. During sampling, the midrib was avoided, and mesophyll tissue from the middle part of the leaves was excised. The samples were flash-frozen in liquid nitrogen and sent to Majorbio Biotechnology Co., Ltd. (Shanghai, China) for transcriptome sequencing using the Illumina NovaSeq 6000 platform (San Diego, CA, USA).

After filtering the raw data to remove low-quality reads, adapter sequences, and contaminated sequences, the clean reads were aligned to the Arabidopsis reference genome (TAIR10) using the HISAT2 software (version 2.2.1) with the default parameters to obtain mapping data. DEG screening was performed using the DESeq2 software (version 1.48.1) with the thresholds of |log₂ fold change| ≥ 1 and p < 0.05. Gene expression levels were quantified based on fragments per kilobase of transcript per million mapped reads (FPKM) values. Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed to acquire functional annotations, biological functions, and related metabolic pathways of all DEGs, as well as to identify key pathways and genes involved in drought stress responses.

4.10. Statistical Analysis

All data were statistically analyzed using the WPS Office software (Jinshan, Beijing, China). Differences were considered statistically significant at p < 0.05. Each experiment included at least three biological replicates, and results are presented as mean ± standard deviation unless otherwise stated.

5. Conclusions

In this study, we explored the function of the SbPIP1-3 gene using three heterologous expression systems—yeast, Arabidopsis, and rapeseed. Our findings systematically demonstrate that this gene enhances drought tolerance in plants through four coordinated mechanisms: First, due to its plasma membrane localization, SbPIP1-3 facilitates efficient transmembrane water transport, helping to maintain cellular water potential and turgor pressure, thereby ensuring basic cellular functions under drought conditions. Second, it upregulates the expression of key genes such as SnRK2, P5CS, and BADH, promoting the synthesis and accumulation of proline and glycine betaine. This enhances cellular dehydration tolerance through osmotic adjustment, macromolecular protection, and antioxidant activity. Third, SbPIP1-3 promotes the expression of antioxidant enzyme genes including SOD, POD, and CAT, establishing an efficient ROS scavenging network that significantly alleviates membrane lipid peroxidation. Fourth, it protects the core photosynthetic machinery from stress-induced impairment, thereby securing its structural and functional integrity. This is achieved through the sustained maximum photochemical efficiency of PSII, coupled with enhanced regulated energy dissipation and non-photochemical quenching, which collectively ensure that fundamental energy production continues under adverse conditions.

This study not only advances the current physiological understanding of how aquaporins contribute to drought resistance, but also offers valuable genetic resources and a theoretical framework for molecular breeding of drought-tolerant crops. Future research should aim to elucidate the cooperative regulatory networks between SbPIP1-3 and other stress resistance pathways, which will pave the way for developing new crop varieties tailored to arid and semi-arid regions.