The apical buds of tissue-cultured seedlings from two potato cultivars with inconsistent genetic backgrounds for heat stress, ‘Atlantic’ (heat-sensitive) and ‘Desiree’ (heat-tolerant), were transferred to MS medium and cultured for 28 days under controlled conditions (16-hour light/8-hour dark photoperiod, 2800 lux light intensity, 20°C). The seedlings were then transplanted into autoclaved substrate soil and cultivated in a growth chamber for 14 days (16-hour light/8-hour dark photoperiod, 2800 lux light intensity, 20°C, 75% relative humidity). Uniform plants were selected and transplanted into pots (26 cm × 27 cm × 18 cm) containing a soil-vermiculite mixture (1:1, v/v) and grown for five weeks under soil moisture maintained at 70–75%. To investigate the response of the StWRKY75 gene to drought, heat, and salt stress, experimental conditions included drought stress induced by 10% and 20% polyethylene glycol 6000 (PEG6000), heat stress at 30°C and 35°C, and salt stress with 75 mM and 150 mM sodium chloride (NaCl). Plants were exposed to these abiotic stresses for 0, 1, 2, 4, 8, 16, 24, and 48 hours, followed by leaf sampling for mRNA expression analysis. To assess StWRKY75 expression levels, 432 potted plants were analyzed, encompassing two potato cultivars, two drought stress treatments (10% and 20% PEG6000), two heat stress treatments (30°C and 35°C), two salt stress treatments (75 mM and 150 mM NaCl), eight time points (0–48 hours), three biological replicates, and three technical replicates (pots). For quantitative evaluation of physiological parameters, photosynthetic indices, and heat-responsive genes under heat stress, 14-day-old transgenic and NT lines of both cultivars from growth chambers were transplanted into pots (26 cm × 27 cm × 18 cm) containing a soil-vermiculite mixture (1:1, v/v), grown for five weeks under 70–75% soil moisture, and subjected to heat stress (30°C and 35°C) for 48 hours. A total of 378 seedlings were evaluated, including two cultivars, seven genotypes per cultivar (one NT line, three StWRKY75 overexpression lines, and three RNAi-mediated silencing lines), two heat treatments (30°C and 35°C), three biological replicates, and three technical replicates.

Multiple-sequence alignment of StWRKY75 with orthologs from 18 additional plant species was conducted using CLUSTALW (version 1.83) (Thompson et al., 1994). Conserved protein motifs were subsequently identified through the same alignment analysis. Phylogenetic reconstruction was performed using MEGA X (version 4.1) (Dereeper et al., 2010), employing the Neighbor-Joining (NJ) method with 1000 bootstrap replicates to assess nodal support.

Total RNA was extracted from the samples using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized from 1 µg of total RNA using the TransScript First-Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). The qRT-PCR was performed on a LightCycler 480 II instrument (Roche, Rotkreuz, Switzerland) in 20 µL reaction mixtures. Each reaction contained 100 ng of cDNA, 0.8 µL of gene-specific primers (see Supplementary Table S1 ), and 10 µL of SYBR Premix Ex Taq (2×) (Takara, Tokyo, Japan). The amplification conditions were as follows: initial denaturation at 94°C for 2 min, followed by 34 cycles of denaturation at 94°C for 30 s, primer annealing at 60°C for 34 s, and extension at 72°C for 30 s. The Stef1α gene was used as an internal control to normalize gene expression levels. Relative gene expression was quantified using the 2^−ΔΔCt method (Livak and Schmittgen, 2001) based on cycle threshold (CT) values. To ensure reproducibility, three biological replicates were analyzed, each with three technical replicates. The primer sequences used in this study are provided in Supplementary Table S1 .

The StWRKY75 coding sequence (GenBank ID: NM_001288675.1) was PCR-amplified using specific primers (forward: 5’-CTCGACATGGAGAATTATGCAACAATAT-3’; reverse: 5’-GTCGACAAAAGGAAGTATAGATTTGCATCT-3’; Bioeditas, Shaanxi, China) and cloned into the pBI121-EGFP vector following established protocols (Li et al., 2020). RNAi lines were generated as previously described (Lu et al., 2019). The sense cDNA fragment was amplified using EcoR I/Kpn I-restriction sites primers and cloned into pHANNIBAL (pHAN-StWRKY75-R). Similarly, the antisense fragment was amplified with Hind III/Xba I primers and inserted to create pHAN-StWRKY75-RF. The pHAN-StWRKY75 construct was then sub-cloned into the pART vector at the Xho I and Xba I restriction sites, resulting in the pART-StWRKY75-RNAi construct. The construct was transformed into Agrobacterium tumefaciens LBA4404 and cultured (28°C, 48 h) for subsequent plant transformation. Transformants were verified by PCR amplification using RNAi-specific primers: 5’-TGGAGAATTATGCAACAATATTTC-3’ (forward) and 5’-CCATCTATAACCATCAAGAAT-3’ (reverse), NPT II selection marker primers: 5’-ATGACTGGGCACAACAGACAATCG-3’ (forward) and 5’-TCAGAAGAACTCGTCAAGAAGGCG-3’ (reverse). The transformed Agrobacterium tumefaciens culture was maintained in an LB medium supplemented with 50 mg/L each of spectinomycin and gentamicin at 28°C for 48 hours. Following centrifugation (5,000 × g, 10 min), bacterial cells were resuspended in MS liquid medium to achieve OD600 = 0.3. Surface-sterilized potato stem segments (2 cm) were cultured on an MS medium containing 7.4 g/L agar, 30 g/L sucrose, 0.5 mg/L 6-benzylaminopurine (6-BA), 2.0 mg/L zeatin (ZT), 0.2 mg/L gibberellic acid(GA3), 1.0 mg/L indole-3-acetic acid (IAA) (pH adjusted to 5.8). The explants were immersed in Agrobacterium suspension for 10 min, then co-cultivated in darkness (72 h, 25°C). Subsequently, samples were transferred to the selection medium (composition as above, supplemented with 300 mg/L timentin, 100 mg/L kanamycin), with media refreshed biweekly. The selection medium was replaced every two weeks to maintain antibiotic pressure. Developing shoots that exhibited stable antibiotic resistance were subsequently excised and transferred to a rooting medium consisting of MS basal salts, 7.4 g/L agar, 30 g/L sucrose, 300 mg/L timentin, and 100 mg/L kanamycin to promote adventitious root formation.

The protein-coding sequence of StWRKY75 was amplified using primers (forward: 5′-ATGGAGAATTATGCAACAATAT-3′; reverse: 5′-AAAAGGAAGTATAGATTTGCATCT-3 ′) and cloned into the pCAM35s-GFP expression vector. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101. Transient transformation of tobacco (Nicotiana benthamiana) epidermal cells was performed following the protocol described by Sparkes et al. (2006). GFP fluorescence was visualized after 48 hours of post-infiltration using a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).

Apical buds of transgenic and NT tissue-cultured seedlings from two uniform potato cultivars, ‘Atlantic’ and ‘Desiree’, were transferred to MS medium and cultured for 28 days under controlled conditions (16-hour light/8-hour dark photoperiod, 2800 lux light intensity, 20°C). The seedlings were then transplanted into autoclaved substrate soil and cultivated in a growth chamber for 14 days (16-hour light/8-hour dark photoperiod, 2800 lux light intensity, 20°C, 70% relative humidity). Uniformly growing plants were selected and transplanted into pots (26 cm × 27 cm × 18 cm) containing a soil-vermiculite mixture (1:1, v/v) and grown for five weeks under soil moisture maintained at 70–75%. Plants with consistent growth were subjected to heat stress (30°C and 35°C) for five weeks, followed by growth parameter analysis. Plant height was measured as the distance from the soil surface to the stem apex, while tuber yield per plant and biomass (fresh and dry weights of shoots and roots) were evaluated under different heat stress conditions. Fresh weights were recorded immediately after treatment, and dry weights were determined after drying samples to constant weight in a 70°C oven. A total of 378 seedlings were assessed, encompassing two cultivars, seven genotypes per cultivar (1 NT line, 3 StWRKY75 overexpression lines, and 3 RNAi-mediated silencing lines), two heat treatments (30°C and 35°C), three biological replicates, and three technical replicates.

The key physiological parameters, including the activities of SOD (Giannopolitis and Ries, 1977), CAT (Aebi, 1984), POD (Maehly and Chance, 1954), and APX (Nakano and Asada, 1981), as well as the levels of proline (Bates et al., 1973) and MDA (Heath and Packer, 1968), were assayed using established protocols ( Supplementary Data 1 ). The study evaluated 378 seedlings from two cultivars, each comprising seven genotypes (an NT control, three StWRKY75-overexpression lines, and three StWRKY75-RNAi lines), under two heat stress regimes (30°C and 35°C) in a completely randomized design.

The photosynthetic capacity of plants was assessed by examining the third fully developed leaf from the shoot apex, with measurements conducted during mid-morning hours (9:30-11:30 AM). Gas exchange measurements, comprising net photosynthesis (Pn), transpiration (E), and stomatal conductance (Gs), were collected using Li-COR’s 6400XT portable photosynthesis monitoring equipment. Measurements were conducted under controlled environmental conditions: photosynthetic photon flux density (PPFD) of 1500 μmol·m⁻²·s⁻¹, atmospheric CO₂ concentration of 400 μmol·mol⁻¹, and leaf chamber relative humidity maintained at 60-70%. The experimental design consisted of three biological replicates arranged in a completely randomized configuration.

The statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, California, USA) and IBM SPSS Statistics 19 (IBM Corporation, New York, USA). Before analysis, data normality was verified using Shapiro-Wilk tests, while homogeneity of variance was confirmed through Levene’s test (P>0.05). All datasets satisfied the assumptions of normality and homoscedasticity. The results are shown as mean ± standard deviation (SD). A two-way analysis of variance (two-way ANOVA) followed by Tukey’s multiple comparisons test was employed to assess differences between groups, with a significance threshold set at P < 0.05.

The amino acid sequence alignment ( Figure 1A ) and phylogenetic tree ( Figure 1B ) demonstrate the evolutionary conservation of StWRKY75 among 18 other plant species. Mango (Mangifera indica) MiWRKY75, tea plant (Camellia sinensis) CsWRKY75, oil-seed camellia (Camellia lanceoleosa) ClWRKY75, Shrub althea (Hibiscus syriacus) HsWRKY75, peruvian cotton (Gossypium raimondii) GrWRKY75, silverweed (Argentina anserina) AaWRKY75, Chinese rose (Rosa chinensis) RcWRKY75, Japanese plum (Prunus mume) PmWRKY75, apple (Malus domestica) MdWRKY75, common pear (Pyrus communis) PcWRKY75, sweet potato (Ipomoea batatas) IbWRKY75, Arabidopsis (Arabidopsis thaliana) AtWRKY75, flowering tobacco (Nicotiana sylvestris) NsWRKY75, wild tobacco (Nicotiana tomentosiformis) NtWRKY75, tomato (Solanum lycopersicum) SlWRKY75, potato (Solanum tuberosum) StWRKY75, ground cherry (Physalis pubescens) PpWRKY75, wolfberry (Lycium barbarum) LbWRKY75, pepper (Capsicum annuum) CaWRKY75 and their predicted amino acid residue alignment is shown in Figure 1A . The NCBI Protein BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) was employed to retrieve the protein accession numbers of WRKY75 orthologs from diverse plant species, as listed in Supplementary Table S1 . The sequence alignment analysis revealed that StWRKY75 shares significant sequence homology with orthologous proteins from diverse species, including Solanum lycopersicum (SlWRKY75), Prunus mume (PmWRKY75), Rosa chinensis (RcWRKY75), Actinidia arguta (AaWRKY75), Gossypium raimondii (GrWRKY75), and Hibiscus syriacus (HsWRKY75). All these proteins contain the conserved signature WRKYGQK at the N-terminus, followed by the C2H2 zinc-finger motif (CX₅-C-X₂₃-H-X₁-H), which is characteristic of group II ( Figure 1A ). This conserved domain architecture confirms the evolutionary preservation of core functional foundations across these orthologs. Phylogenetic analysis revealed distinct clustering patterns among WRKY75 orthologs ( Figure 1B ). Notably, StWRKY75 exhibits the highest sequence homology with SlWRKY75, as they are clustered together in the phylogenetic tree. Members within each cluster exhibit conserved functional domains and significant evolutionary homology, suggesting conserved functional roles among phylogenetically related orthologs.

To elucidate the transcriptional dynamics of StWRKY75 under various abiotic stresses, this study systematically analyzed the relative expression patterns in leaves of potato cultivars ‘Desiree’ and ‘Atlantic’ using qRT-PCR ( Figures 2 , 3 ). In ‘Desiree’, StWRKY75 expression showed continuous upregulation from 1 to 48 hours post-treatment (hpt) under 30°C (peaking at 48 hpt), while 35°C stress treatment induced a sustained upregulated expression pattern reaching maximum at 48 hpt ( Figures 3C, D ). ‘Atlantic’ displayed significant upregulation from 1–48 hpt under 30°C (peak at 2 hpt), with 35°C stress treatment triggering rapid response (peak at 4 hpt), indicating the gene’s involvement in thermotolerance regulation through rapid response to heat stress ( Figures 2C, D ). Under drought stress, ‘Desiree’ showed insensitivity to 10% PEG6000 (only significant downregulation at 1 hpt), whereas 20% PEG6000 treatment caused continuous suppression from 1–48 hpt (minimum at 16 hpt), suggesting its potential negative regulatory role in severe drought response ( Figures 3A, B ). ‘Atlantic’ exhibited significant upregulation at 4–8 hpt (peaks at 4 and 8 hpt) but downregulation at other time points (minimum at 1 hpt) under 10% PEG6000, with 20% PEG6000 inducing overall suppression (minimum at 48 hpt) ( Figures 2A, B ). Salt stress responses showed cultivar-specific patterns: ‘Desiree’ showed no significant changes under 75 mM NaCl but significant upregulation at 4 hpt (peak at 48 hpt) under 150 mM NaCl ( Figures 3E, F ); ‘Atlantic’ displayed only transient upregulation at 16 hpt under 75 mM NaCl, while 150 mM NaCl caused significant suppression from 4–16 hpt (minimum at 8 hpt) ( Figures 2E, F ). These findings demonstrate that StWRKY75 functions as a core regulator in heat stress response with rapid activation characteristics under both 30°C and 35°C stress treatments, while its roles in drought and salt stress may be differentially modulated by cultivar-specific genetic backgrounds and stress intensity.

To assess the subcellular localization of StWRKY75 TF, tobacco leaves were infiltrated with Agrobacterium tumefaciens strain GV3101 carrying the recombinant plasmid pCAM35S-GFP-StWRKY75 and the empty vector control (pCAM35S-GFP). Laser confocal microscopy showed that the StWRKY75-GFP fusion protein localized exclusively to the nucleus by green protein signals ( Figure 4 ). In contrast, the GFP vector (control) was distributed in the plasma membrane, cytoplasm, and nucleus. These results demonstrate the nuclear-specific localization of StWRKY75.

To functionally characterize StWRKY75 in potato, we established transgenic overexpression (OE) and RNA interference (RNAi) lines in two commercial cultivars, ‘Atlantic’ and ‘Desiree’. The qRT-PCR analysis revealed that StWRKY75 transcript levels were significantly elevated in all OE lines (OE-1 to OE-8; *** P < 0.001, Supplementary Figure S1 ), whereas its expression was markedly suppressed in RNAi lines (RNAi-1 to RNAi-8; * * *P < 0.001, Supplementary Figure S1 ). Based on expression profiling, we selected three most representative lines from each group for subsequent functional studies: OE-3, OE-6, and OE-7 (showing highest overexpression) along with RNAi-1, RNAi-4, and RNAi-5 (exhibiting strongest suppression) in ‘Atlantic’(* * *P < 0.001, Supplementary Figures S1A, B ); similarly, OE-1, OE-2, OE-4 and RNAi-1, RNAi-3, RNAi-4 were chosen from ‘Desiree’ lines (* * *P < 0.001, Supplementary Figures S1C, D ). These selected transgenic lines were subsequently employed to investigate the functional role of StWRKY75 in thermotolerance responses.

Under optimal growth conditions (20°C), transgenic and NT control lines of potato cultivars ‘Atlantic’ and ‘Desiree’ exhibited comparable performance (P>0.05) in all evaluated growth parameters, including plant height, tuber weight per plant, fresh weight, dry weight, root fresh weight, and root dry weight ( Table 1 ). When exposed to 30°C heat stress, all genotypes of both cultivars exhibited significant reductions in growth metrics (*P<0.05, **P<0.01). However, StWRKY75-overexpressing lines in both ‘Atlantic’ and ‘ Desiree’ demonstrated enhanced thermotolerance, with significantly greater plant height, tuber yield, and biomass accumulation compared to NT and RNAi lines ( Table 1 ). Conversely, RNAi lines showed pronounced growth inhibition, with all measured parameters significantly lower than NT controls (*P<0.05, **P<0.01). At 35°C, heat stress, growth parameters declined more severely across all genotypes, with heat stress severely impeding potato growth. Nevertheless, OE lines in both cultivars maintained significantly superior growth performance ( Table 1 ), while RNAi lines suffered exacerbated heat stress damage, displaying further significant reductions in biomass and tuber yield compared to NT plants (*P<0.05, **P<0.01, ***P<0.001). These findings collectively indicate that StWRKY75 overexpression confers thermotolerance by mitigating heat-induced growth suppression, while its knockdown exacerbates sensitivity, underscoring the gene’s critical role in potato heat stress adaptation.

To assess the hypothesis that StWRKY75-overexpressed potato plants of cultivars ‘Atlantic’ and ‘Desiree’ regulate antioxidant metabolism under heat stress, the activities of key antioxidant enzymes, including APX, CAT, POD, and SOD along with the oxidative stress marker MDA and the non-enzymatic osmolyte proline were investigated under varying temperature stress treatments (20°C, 30°C, and 35°C). Under control conditions (20°C), no significant differences were observed in APX, CAT, POD, or SOD activities, MDA levels, or proline content between transgenic (StWRKY75-overexpressing and RNAi) and NT plants of the potato cultivar ‘Atlantic’ ( Figures 5A-F ). However, when subjected to 30°C heat stress, the StWRKY75-overexpressing lines (OE-3, OE-6, OE-7) exhibited significantly enhanced activities of APX ( Figure 5A ), CAT ( Figure 5B ), POD ( Figure 5C ), and SOD ( Figure 5D ), along with increased proline accumulation ( Figure 5F ). In contrast, RNAi knockdown lines (RNAi-1, RNAi-4, RNAi-5) showed marked reductions in these parameters compared to NT controls. Notably, MDA content, a key indicator of oxidative damage, was significantly lower in StWRKY75-overexpressing plants ( Figure 5E ), whereas RNAi lines accumulated higher MDA levels, indicating greater susceptibility to oxidative stress. Under more severe heat stress (35°C), StWRKY75-overexpressing plants showed significantly greater increases in the activities of APX ( Figure 5A ), CAT ( Figure 5B ), POD ( Figure 5C ), and SOD ( Figure 5D ) activities, along with significantly elevated proline content ( Figure 5E ), when compared with NT control plants. Conversely, RNAi lines exhibited further suppression of these physiological indices. The StWRKY75-overexpressing lines maintained lower MDA accumulation ( Figure 5F ) at 35°C, demonstrating reduced oxidative damage, while RNAi lines showed exacerbated membrane lipid peroxidation due to impaired stress tolerance.

Similarly, the cultivar ‘Desiree’ displayed comparable responses under high-temperature stress treatments (30°C and 35°C). Under control conditions (20°C), no significant differences were observed among all experimental potato plants for different physiological parameters ( Figures 6A-F ). However, when heat stress treatment was increased (30°C), the StWRKY75-overexpressing lines (OE-1, OE-2, OE-4) exhibited significantly increased activities of APX ( Figure 6A ), CAT ( Figure 6B ), POD ( Figure 6C ), and SOD ( Figure 6D ), and higher proline accumulation ( Figure 6F ). In contrast, RNAi-silenced lines (RNAi-1, RNAi-3, RNAi-4) showed a pronounced reduction in these physiological markers. Additionally, the MDA content was significantly lower in StWRKY75-overexpressing lines ( Figure 6E ), whereas RNAi lines exhibited higher MDA levels compared to NT control lines. Under severe heat stress conditions (35°C), all experimental lines exhibited exacerbated physiological damage. However, the StWRKY75-overexpressing line demonstrated significantly enhanced enzymatic activity of key antioxidant enzymes, including APX ( Figure 6A ), CAT ( Figure 6B ), POD ( Figure 6C ), and SOD ( Figure 6D ), as well as elevated proline level ( Figure 6F ). In contrast, StWRKY75 RNA interference (RNAi) lines exhibited a noticeable decline in these physiological parameters compared to NT plants. Likewise, the StWRKY75-overexpressing lines showed declined MDA levels ( Figure 6F ), and the opposite was true for StWRKY75-knockdown lines than those in NT potato lines. These findings suggest that StWRKY75 plays a critical regulatory role in enhancing thermotolerance by modulating antioxidant defense mechanisms and osmoprotectant synthesis under extreme heat stress conditions.

To investigate the genetic regulatory networks and biochemical mechanisms underlying heat stress adaptation in two distinct potato cultivars, ‘Atlantic’ and ‘Desiree’, this study systematically analyzed the expression profiles of key stress-responsive genes (including StAPX, StCAT, StSOD, StPOD, StP5CS, StHSP20, StHSP70, and StHSP90) across all transgenic and NT lines under different heat stress treatments (20°C, 30°C, and 35°C). The research aimed to comprehensively evaluate the differential regulation of antioxidant enzyme-related genes, proline biosynthesis-related genes, and heat shock-related genes under escalating temperature stress, thereby elucidating the genetic basis of StWRKY75-mediated thermotolerance.

Under control conditions (20°C), no significant differential expression was observed for any of the assessed stress-responsive genes (StAPX, StCAT, StSOD, StPOD, StP5CS, StHSP20, StHSP70, and StHSP90) in ‘Atlantic’ potato plants ( Figures 7A-H ). However, when temperature was increased to 30°C, StWRKY75-overexpressing plants (OE-3, OE-6, OE-7) showed significant upregulation in transcript levels of StAPX ( Figure 7A ), StCAT ( Figure 7B ), StSOD ( Figure 7C ), StPOD ( Figure 7D ), StP5CS ( Figure 7E ), StHSP20 ( Figure 7F ), StHSP70 ( Figure 7G ), and StHSP90 ( Figure 7H ), while StWRKY75 RNA interference lines (RNAi-1, RNAi-4, RNAi-5) exhibited marked downregulation compared to NT controls. This differential expression pattern became more pronounced under 35°C heat stress, with StWRKY75-overexpressing lines showing extremely significant upregulation of these stress-responsive genes relative to NT plants. Conversely, all target gene expressions were suppressed in StWRKY75 knockdown plants. Similarly, under standard growth conditions (20°C), the ‘Desiree’ cultivar maintained stable transcriptional levels of these key stress-responsive genes across NT and transgenic lines without significant differences ( Figures 8A-H ). At 30°C heat stress, however, StWRKY75-overexpressing lines (OE-1, OE-2, OE-4) demonstrated significant upregulation of antioxidant genes [StAPX ( Figure 8A ), StCAT ( Figure 7B ), StSOD ( Figure 7C ), StPOD ( Figure 8D )], the proline biosynthesis gene StP5CS ( Figure 8E ), and heat shock protein genes [StHSP20 ( Figure 8F ), StHSP70 ( Figure 8G ), StHSP90 ( Figure 8H )]. In contrast, these stress-responsive genes were significantly suppressed in StWRKY75 knockdown lines (RNAi-1, RNAi-3, RNAi-4) compared to NT plants. Elevated temperatures intensified these transcriptional responses, with the most prominent differential expression observed at 35°C. At this threshold, StWRKY75-overexpressing plants maintained strong upregulation of all stress-related genes ( Figures 8A-H ), while RNAi lines showed progressively declining expression profiles. These findings robustly demonstrate that StWRKY75 functions as a central transcriptional regulator during thermal adaptation, coordinating the upregulation of these genes (StAPX, StCAT, StSOD, StPOD, StP5CS, StHSP20, StHSP70, and StHSP90) to enhance potato thermotolerance.

Under high-temperature stress regimes (30°C and 35°C), key photosynthetic parameters, including Pn, Gs, and E, were measured. Under control conditions (20°C), no significant differences were observed in gas exchange parameters between transgenic and NT plants of both ‘Atlantic’ and ‘Desiree’ potato cultivars ( Figures 9A-F ). However, at 30°C, StWRKY75-overexpressing lines in both cultivars exhibited significantly enhanced photosynthetic rate ( Figures 9A, B ), transpiration rate ( Figures 9C, D ), and stomatal conductance ( Figures 9E, F ) compared to NT plants. Conversely, StWRKY75-knockdown lines showed reduced photosynthetic performance. Under severe heat stress (35°C), growth inhibition was observed in all potato plants due to thermal damage. Nevertheless, StWRKY75-overexpressing lines maintained significantly higher photosynthetic rate ( Figures 9A, B ), transpiration rate ( Figures 9C, D ), and stomatal conductance ( Figures 9E, F ) compared to NT controls in both cultivars. In contrast, StWRKY75-knockdown plants displayed markedly reduced gas exchange parameters relative to NT lines, demonstrating the crucial regulatory role of StWRKY75 in maintaining photosynthetic performance under heat stress.