Cloning and overexpression of the DaSOD1 gene from Dioscorea alata improves cadmium resistance in transgenic tobacco plants

ABSTRACT

Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar characterized by its strong resistance to Cd stress. This study aims to investigate the enzymatic antioxidant system of MH1 under Cd stress. Under Cd treatment, MH1 exhibited an elevation in superoxide dismutase (SOD) activity, while the activities of ascorbate peroxidase, catalase, and peroxidase remained unchanged. A Cu/Zn-SOD gene, DaSOD1, was cloned and found to be up-regulated following Cd treatment. The DaSOD1 gene was further introduced into tobacco plants for functional analysis. Transgenic tobacco seedlings overexpressing DaSOD1 under Cd treatments exhibited increased chlorophyll contents and seed germination rate, while the levels of superoxide anion and malondialdehyde decreased compared to the wild-type plants. This suggests that DaSOD1 overexpression mitigated the negative effects of Cd stress by reducing oxidative damage in plants. Characterization of the SOD activity and its corresponding DaSOD1 gene is expected to improve our understanding of Cd-resistance mechanism in yam plants.

KEYWORDS:

• Cadmium
• Dioscorea alata
• oxidative stress
• superoxide dismutase
• transgenic plants

Abbreviations

APX:	=	ascorbate peroxidase
Chl:	=	chlorophyll
CAT:	=	catalase
MDA:	=	malondialdehyde
MH1:	=	Minghuai 1
MS:	=	Murashige and Skoog
POD:	=	peroxidase
SOD:	=	superoxide dismutase
WT:	=	wild-type

Introduction

Cadmium (Cd) pollution is recognized as one of the most extremely toxic heavy metals in soils, primarily resulting from industrial processes and the use of phosphate fertilizers. Cd pollution has become a significant global environmental concern due to its non-degradable nature, posing a harmful threat to plant growth and food safety (Clemens et al. 2013; El Rasafi et al. 2022). In the realm of agriculture, the long-term overuse and abuse of certain fertilizers and pesticides containing Cd can result in the release of Cd into the atmosphere, water, and soil (Cheraghi et al. 2012; Zwolak et al. 2019). This heavy metal contaminates the soil, and can be absorbed and accumulated by plants, leading to harmful damage. Cd stress results in the inhibition of seed germination, root elongation, seedling growth, water and nutritional imbalance, plant biomass, and productivity by alternating various physiological processes in plants, such as photosynthetic efficiency, relative water content, transpiration rate, and stomatal conductance (El Rasafi et al. 2022; García de la Torre et al. 2022). It is worth noting that Cd can be phytotoxic even at low concentrations (Choppala et al. 2014). These physiological disorders caused by Cd toxicity are believed to be correlated with the overproduction of reactive oxygen species (ROS), including hydroxyl radical (OH^-), hydrogen peroxide (H₂O₂), and superoxide radicals (O₂⁻), leading to oxidative damages of DNA, proteins, and membrane lipids in plant cells (Guan et al. 2009; Meng et al. 2019; Moradi et al. 2019; Khan et al. 2022).

To metabolize the harmful effects of Cd-induced oxidative stress in plant cell, plants have evolved sophisticated antioxidative defense mechanisms, including both enzymatic and non-enzymatic antioxidants (Guan et al. 2009; Li et al. 2017; El Rasafi et al. 2022). Recently, many studies reported that enzymatic antioxidant defenses are crucial for plant resistance to Cd exposure. Ascorbate peroxidase (APX), catalase (CAT), glutathione-related enzymes, peroxidase (POD), and superoxide dismutase (SOD) in plants have been widely reported to be upregulated in response to Cd exposure (Chen et al. 2022). Notably, different plant species exhibit distinct responses in terms of antioxidant enzyme activities under Cd stress (Meng et al. 2019). In rice seedlings, high levels of Cd enhanced activities of APX, monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), glutathione peroxidase (GPX), and SOD, whereas the activities of catalase (CAT) was attenuated (Rahman et al. 2016). In response to Cd conditions, the activities of APX, CAT, and SOD were induced in both cucumber and hollyhocks (Althea rosea) (Semida et al. 2018). Meng et al. (2019) reported that the activities of CAT, POD, and SOD were decreased in lettuce roots under Cd treatments, while the activities of these antioxidant enzymes remained steady or showed enhancement in the roots of spinach and garland chrysanthemum. Moreover, another study suggested that SOD and POD serve as critical defense enzymes in wheat, primarily due to their elevated activities under Cd treatment (Qin et al. 2022). These findings suggest that the relationship between antioxidant enzyme activation and Cd resistance can be sophisticated and may vary among different plant species.

Several strategies are developed to manage Cd pollution, such as phytoremediation through the cultivation of Cd-tolerant plant cultivars or hyper-accumulator plant species (Guan et al. 2009; Patel et al. 2022). In recent decades, significant research efforts have been focused on the utilization of genetically modified plants. These plants express resistance genes, offering an effective, low-cost, and eco-friendly method for phytoremediation of Cd pollution (Guan et al. 2009; Li et al. 2017; García de la Torre et al. 2022). Hence, the identification of resistance genes from Cd-tolerant plants holds significant importance. To date, numerous resistance genes that contribute to elevated antioxidant enzyme activity against Cd toxicity have been cloned and characterized in various Cd-tolerant plants (Guan et al. 2009; Mahmoud et al. 2021; Zhang et al. 2023). Yam (Dioscorea alata L.) is an economically important food crop and traditionally medicinal crop in Asia and Africa, known for its nutritional and medicinal properties, including carbohydrate, minerals, dietary fiber, vitamin C, anthocyanin, and diosgenin (Olawale et al. 2023). The yam Minghuai 1 (MH1), bred by the Sanming Academy of Agricultural Sciences (Fujian, China), is a purple-flesh yam cultivar that exhibits strong resistance to various environmental challenges, such as flooding, drought, and pathogen infections (Chen et al. 2019; Hua et al. 2021). Previous reports have indicated that MH1 can activate a robust enzymatic antioxidant defense, contributing to its resistance against abiotic stressors. (Chen et al. 2019). Exposure to Cd stress can result in a range of physiological changes in plants, particularly the over-accumulation of ROS. Nevertheless, the ROS-scavenging capability and physiological responses of yam plants under Cd stress have received limited attention. Therefore, comprehending how yam plants mitigate Cd-induced oxidative damage at both the physiological and molecular levels is essential for developing strategies to enhance yam production.

In this study, the yam MH1 cultivar drew our attention due to its high resistance to Cd stress. We examined the antioxidant enzyme activities, including APX, CAT, POD, and SOD, in MH1 subjected to Cd treatments. Furthermore, a Cd-induced CuZnSOD gene was cloned and employed to generate transgenic tobacco plants for functional gene analysis. The diminished Cd-induced damages observed in transgenic tobacco plants overexpressing the DaSOD1 gene suggest that the presence of the DaSOD1 gene benefits the improvement of Cd resistance in transgenic plants. More importantly, our findings contribute to a genetic engineering approach for phytoremediation, involving the prompt scavenging of Cd-induced excessive ROS in plant cells through the overexpression of DaSOD1.

Materials and methods

Plant materials and stress treatments

Yam MH1 and MH3 cultivars were bred by the Sanming Academy of Agricultural Science. The yam seedlings were planted in 3-inch plastic pots filled with a 3:1:1 potting mixture of peat, vermiculite and perlite in a phytotron at 25°C/20°C (light/dark) with a 16-hour photoperiod (100 μmol m⁻² s⁻¹). Thirty-day-old uniform and robust yam seedlings were treated with 0, 50, 200 μM cadmium chloride (CdCl₂) for 7, 14, 21, and 28 days. In the mock treatment, the plants were treated with distilled-deionized (dd) water for 7, 14, 21, and 28 days. Leaves and stems of seedlings from each sample were harvested at the indicated times and quickly frozen by liquid nitrogen for physiological, antioxidant, and molecular analyses.

Tobacco (Nicotiana benthamiana) seeds were sterilized in 1% (v/v) NaOCl solution for 10 min and then seeded on 1/2 Murashige and Skoog (MS) agar medium supplemented with 3% sucrose. After germination, seven-day-old wild-type (WT) and transgenic tobacco seedlings were transferred to 1/2 MS agar medium containing 0, 100, and 200 μM CdCl₂10 days in a growth chamber at 25°C/20 °C (light/dark) with a 16-hour photoperiod (100 μmol m⁻² s⁻¹). At specified time intervals, the seedlings were harvested by using liquid nitrogen and stored at −80 °C for further analysis. To evaluate the seed germination rate assay, the seeds with sterilization were plated on 1/2 MS agar medium containing 0, 100, and 200 μM CdCl₂ for 4, 6, and 8 days.

Assays for Cd accumulation in MH1 and MH3

Thirty-day-old uniform and robust yam seedlings were treated with or without 200 μM CdCl₂ for 21 days. The leaves and stems of seedlings from each sample were harvested and quickly frozen by liquid nitrogen. For Cd determination, Inductively Coupled Plasma Mass Spectroscopy analysis (ICP-MS, the iCAP Q ICP-MS, Thermo Fisher Scientific, USA) was used following standard methods (GB5009.268-2016) of national food safety standards of China (Sanming Laboratory of the Bureau of Geology and Mineral Exploration in Fujian).

Assays for physiological parameters and antioxidant enzyme activities

The relative chlorophyll content of MH1 and MH3 plants was assessed using a SPAD analyzer (SPAD-502 Chlorophyll Meter, Konica Minolta, Tokyo, Japan), while the plant photochemical efficiency of photosystem II (Fv/Fm) was determined using a MiniPAMII analyzer (Walz, Effeltrich, Germany). In addition, the chlorophyll a (Chl a) and chlorophyll b (Chl b) in transgenic tobacco plants were conducted according to the method described by Wintermans and De Mots (1965).

For determination of APX (EC1.11.1.11), CAT (EC 1.11.1.6), and POD (EC 1.11.1.7) of MH1 plants under CdCl₂ treatments, the leaf powder (0.1 g) was extracted with 100 mM sodium phosphate buffer (pH 6.8) with ice-cold 1-mM phenylmethylsulfonyl fluoride (Sigma, USA). The extracts were subsequently analyzed for APX, CAT, and POD activities using the methods previously outlined by Nakano and Asada (1981), Hwang and VanToai (1991), and MacAdam et al. (1992), respectively. For CAT activity analysis, 100 μl of leaf extract was incubated with 50 mM potassium phosphate buffer (pH 7.0) and 15 mM H₂O₂. The activity was determined by measuring the changes in absorbance at 240 nm in 1 min using an Infinite M200 plate reader (Tecan, Switzerland). For POD activity analysis, the leaf extract (100 μl) was mixed with 50 mM potassium phosphate buffer (pH 5.8), 21.6 mM guaiacol, and 39 mM H₂O₂, and the activity was determined at 470 nm using an Infinite M200 plate reader (Tecan, Switzerland). The APX activity in the yam plants was assessed by measuring absorption at 290 nm using an Infinite M200 plate reader (Tecan, Switzerland). The activity was calculated as a decline over 1 min at 290 nm absorbance reading in a reaction mixture containing 1 mM H₂O₂ to a mixture consisting of 50 mM potassium phosphate buffer (pH 7), 0.1 mM EDTA, 1.5 mM ascorbate, and 100 μl of the enzyme solution. For the measurement of SOD activity (EC 1.15.1.1), the extracts were assessed using the SOD Assay Kit (Solarbio, China), followed by determination of absorbance at 560 nm using an Infinite M200 plate reader (Tecan, Switzerland).

RNA extraction and gene expression analysis

Total RNA was extracted from 0.1 g of frozen leaf powder using the pine tree method, as previously described by Chang et al. (1993). For the synthesis of complementary (c) DNA, 2 μg of total RNA was employed, and the process was carried out using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan).

The real-time quantitative PCR preparations consisted of one cycle at 95°C for 30 s, followed by 45 cycles of 95°C for 5 s and 60°C for 20 s, using the ABI QuantStudio 3 (USA). The relative gene expression levels were calibrated using the 2^−Δct and 2^−ΔΔct method, employing gene-specific primers (detail in Table S1) for the amplification of the DaSOD1, DaActin, and NbActin genes.

Gene construction and plant transformation

The coding region of DaSOD1 was amplified using the gene specific primers (Table S1) from the cDNA of MH1 plants in the CdCl₂ treatment. The PCR product was then cloned into pCAMBIA1300-35S-nos binary vector (Chen et al. 2016) for plant gene expression by BamHI and XmaI restriction enzymes. The constructed pCAMBIA1300-35S-DaSOD1-nos plasmid was transferred into Agrobacterium tumefaciens strain GV3101 according to the freeze–thaw method (Wise et al. 2006). Leaf disks from tobacco plants subjected to transformation. The transformed plants were selected and regenerated on MS (Murashige and Skoog Stock) medium supplemented with 20 ppm hygromycin using standard transformation methods.

To validate the presence of DaSOD1 gene in transgenic tobacco plants, the genomic DNA, which was extracted from transgenic plants using the cetyltrimethylammonium bromide method, was analyzed and verified by PCR amplification of the hygromycin phosphotransferase II (HptII) gene. The predicted sizes of HptII gene (297 bp) were confirmed and separated by gel electrophoresis on a 1.5% agarose gel.

Measurements of malondialdehyde (MDA), superoxide radicals, and root cell death

The leaves of transgenic tobacco plants were ground into fine powder using liquid nitrogen. The powdered leaves were then extracted and analyzed using the MDA assay kit (Solarbio, Beijing, China) at 450, 532, and 600 nm, following the manufacturer’s instructions for MDA analysis. To determine the superoxide radicals in the transgenic plants, the Superoxide Anion Activity Content Assay Kit (Solarbio, Beijing, China) was utilized. Following the manufacturer’s instructions, the superoxide radicals reacted with hydroxylamine hydrochloride to generate nitrite. Subsequently, the products were reacted with sulfanilamide and N-(1-naphthyl) ethylenediamine, forming a purple-red azo compound with absorption at 530 nm for quantification of superoxide radicals.

Cell viability was estimated using the trypan blue dye exclusion assay. Following treatment with 200 μM CdCl₂ for 10 days, the roots of both WT and DaSOD1-overexcpressing plants were immersed in a 0.3% trypan blue solution (Sigma, USA). After 20 min, the roots were rinsed with ddwater for five times to remove any excess dye before observation under the Olympus SZX10 microscope.

Phylogenetic analysis

Alignment of 31 SOD protein sequences from different plants was accomplished using the Clustal-W software (Thompson et al. 1997). For phylogenetic analysis, the MEGA 4.0 software was employed, and the Neighbor Joining (NJ), minimum evolution (ME), and maximum parsimony (MP) methods were utilized to generate a tree to study the evolutionary relationships. Significance of clustering was evaluated through bootstrap analysis with 1000 replications (Rzhetsky and Nei 1992; Tamura et al. 2007).

Statistical analysis

All experiments in this research were conducted with a minimum of three biological replicates, as indicated in the legends of each table and figure. The experimental design followed a completely randomized approach. The statistical significance of physiological parameters and antioxidant enzyme activities in MH1 plants subjected to various CdCl₂ concentrations were determined by the one-way analysis of variance (ANOVA), accompanied with a least significant difference (LSD) test at a significance level of P < 0.05 using the Predictive Analytics SoftWare (PASW) Statistics 18 software (IBM Corp., Armonk, NY, USA). Gene expression levels, root inhibition, seed germination rate, and antioxidant properties were statistically compared between the transgenic tobacco plants and WT plants by a two-tailed Student’s t-test at significance levels of P < 0.05 and P < 0.01 using Excel 2018 software.

Results

MH1, a cadmium resistant yam cultivar

The seedlings of MH1 and MH3 were subjected to different concentrations of CdCl₂ (0, 50, 200 μM) for 7, 14, 21, and 28 days. Physiological characteristics, including chlorophyll content (SPAD) and chlorophyll fluorescence (Fv/Fm), were examined in the treated plants to determine whether MH1 exhibited stronger resistance to Cd stress. After the treatments, almost no significant differences were observed in the SPAD and Fv/Fm values between the CdCl₂-treated and mock-treated MH1 plants (Figure 1a and c), while SPAD and Fv/Fm values in CdCl₂-treated MH3 plants were significantly lower than those in mock-treated MH3 plants at 14 and 28 days of CdCl₂ treatments (Figure 1b and d). Accordingly, these results suggest that MH1 is a Cd-tolerant yam cultivar. In addition, the accumulation of Cd in aboveground tissues of MH1 and MH3 plants was determined after exposure to 200 μM CdCl₂ for 21 days, and no statistical difference in Cd accumulation was observed between MH1 and MH3 (Figure S1).

Figure 1. Effects of cadmium stress on physiological parameters, including SPAD and Fv/Fm values, in leaves of yam cultivar MH1 (a, c) and MH3 (b, d), respectively. Data are the means (n = 5) with corresponding standard deviations. Different letters indicate significant differences determined by the one-way ANOVA (P < 0.05).

Antioxidant enzyme activity analysis in MH1 under cadmium treatment

In order to study the defense mechanisms of Cd resistance in MH1 plants, we further monitored the enzymatic ROS scavengers, including APX, CAT, POD, and SOD, between MH1 and MH3 plants subjected to Cd stress. In MH1 plants subjected to CdCl₂ treatments, the activities of APX, CAT, and POD did not show significant differences compared to those in mock-treated plants, except for a slight decrease in CAT activity under 50 μM of CdCl₂ treatment (Figure 2a–c). In contrast, significantly higher levels of SOD activity were observed in MH1 plants treated with CdCl₂. Under the treatment of 200 μM CdCl₂, the activity of SOD in MH1 was increased by 2.65-fold, 2.12-fold, and 2.49-fold at 24 h, 14, and 28 days, respectively, compared to the mock treatment, suggesting that SOD may be the key enzyme contributing to resistance against Cd stress in MH1 (Figure 2d). However, the SOD activity did not change in response to the 50 μM CdCl₂ treatment. In MH3 plants subjected to CdCl₂ treatments, a reduction in APX activity was observed at 24 h and 7 days after treatment, while a decline in CAT activity was noted at 24 h and 28 days post-treatment (Figure 2a and b). In contrast, the activities of POD and SOD were increased after 50 and 200 μM CdCl₂ treatments for 7 and 14 days, respectively (Figure 2c and d). Notably, significantly higher SOD activity was detected in MH1 plants compared to MH3 plants under 200 μM CdCl₂ treatment (Figure 2d), while higher APX activity was observed only at 7 days of 200 μM CdCl₂ treatment in MH1 plants compared to MH3 plants (Figure 2a). However, there was no significant change in CAT and POD activity at any treatment time points between MH1 and MH3 (Figure 2b and c).

Figure 2. Effects of cadmium treatments on antioxidant enzyme activity in MH1 and MH3 cultivars. The activities of APX (a), CAT (b), POD (c), and SOD (d) were detected in the leaves of MH1 and MH3 plants exposed to mock (0 μM), 50 μM, 200 μM CdCl₂ treatments for 24 h, 7, 14, and 28 d. Data are the means (n ≥ 4) with corresponding standard deviations. Different letters indicate significant differences among CdCl₂ treatments within each cultivar determined by the one-way ANOVA (P < 0.05). An asterisk indicates a significant difference between MH1 and MH3 plants subjected to each treatment as determined by Student’s t-test (P < 0.05).

Gene expression analysis and cloning of the DaSOD1 gene

To identify the genes contributing to elevated SOD activity in MH1 under Cd stress, the expressions of six SOD genes, selected from the transcriptomic dataset of MH1 (NCBI SRA accession PRJNA544814.), were analyzed under 200 μM CdCl₂ treatment. After 21 and 28 days of Cd exposure, no significant changes (P > 0.05) were observed in the expressions of the genes TR8249_c0_g1, TR45043_c0_g1, and TR68390_c0_g1 between the CdCl₂-treated plants and the mock-treated plants (Figure 3). In contrast, the expression levels of the TR51994_c0_g1 and TR44896_c0_g1 genes in MH1 were significantly increased (P < 0.05) after 28 days of CdCl₂ treatment. Notably, only the TR51994_c0_g1 gene was induced under both 21 and 28 days of CdCl₂ treatments.

Figure 3. The expression analysis of the superoxide dismutase genes in MH1 cultivar exposed to mock and 200 μM CdCl₂ cadmium treatment for 21 and 28 d. Relative amounts were calculated with respect to DaActin gene, and fold changes of expression levels were calibrated by the mock-treated plants. Data are the means (n = 5) with corresponding standard deviations. Statistical significance between the mock-treated plants and cadmium-treated plants was determined by Student’s t-test (*P < 0.05; **P < 0.01).

The coding region of the TR51994_c0_g1 gene was cloned from the Cd-treated MH1 plants. The TR51994_c0_g1 gene encodes a protein of 152 amino acids with a calculated molecular mass of 15.17 kDa and an isoelectric point of 5.47 analyzed by using the ExPASy site (http://www.expasy.ch/tools/protparam.html). Furthermore, the protein, which contains four conserved Cu²⁺ binding sites and four conserved Zn²⁺ binding sites (Figure S2), was predicted to be localized in the cytoplasm using the PSORT Prediction (https://www.genscript.com /psort.html). Based on the in silicon analyses, the TR51994_c0_g1 gene was renamed as DaSOD1 gene. Figure 4 presents a phylogenetic analysis of the amino acid sequences of 31 SOD genes in plants. These sequences were divided into three clades based on their sequence similarity, including iron-containing superoxide dismutase (Fe-SOD), copper/zinc-containing superoxide dismutase (Cu/Zn-SOD), and manganese-containing superoxide dismutase (Mn-SOD). The DaSOD1 protein was clustered in the Cu/ZnSOD group and exhibited a sequence identity of 92% to the Cu/ZnSOD protein of Litchi chinensis.

Figure 4. Phylogenetic analysis of the DaSOD1 protein and other SOD proteins from various plant species. The 31 amino sequences of plant SOD genes were retrieved from NCBI GenBank database as follows: HsCu/ZnSOD (XP_038990856.1), SaCu/ZnSOD (AAL85888.1), LcCu/ZnSOD (ABY65355.1), PdCu/ZnSOD (XP_008796193.1), SmCu/ZnSOD (APT43083.1), RaCu/Zn SOD (XP_018435956.1), EcCu/ZnSOD (ATL76060.1), MeCu/ZnSOD (XP_021621658.1), SoCu/ZnSOD (XP_030467015.1), AcCu/ZnSOD (XP_020103520.1), NtoCu/ZnSOD (XP_009624899.1), SlCu/ZnSOD (NP_001234031.2), AtCu/ZnSOD (NP_001077494.1), PgCu/ZnSOD (XP_031405382.1), CqCu/ZnSOD (XP_021773039.1), SmaCu/ZnSOD (AJE26130.1), PaCu/ZnSOD (XP_034917943.1), RsFeSOD (XP_018476913.1), PmFeSOD (XP_008234026.1), SlFeSOD (NP_001300698.1), BnaFeSOD (NP_001302710.1), GhFeSOD (NP_001385448.1), TaFeSOD (AFV08636.1), GmMnSOD (NP_001235066.2), PjMnSOD (GFP86610.1), AaMnSOD (PWA75887.1), MaMnSOD (AED99253.1), RpMnSOD (QWT72338.1), LcMnSOD (AGA16522.1), and BjMnSOD (AEB00557.1). Phylogenetic relationships were analyzed by the Neighbor-joining method, and a bootstrap analysis with 1000 replications was calculated using MEGA4 software. Three different strips indicted the different groups of SOD protein including Cu/Zn-SOD, Fe-SOD, and MeSOD.

Overexpression of the DaSOD1 gene in transgenic tobacco plants

To determine the biological function of DaSOD1 gene in resistance against Cd stress, transgenic tobacco plants overexpressing DaSOD1 gene were generated. Five independent transgenic lines (DaSOD-1 to −5), were obtained and confirmed by genomic DNA PCR amplification. All the DaSOD1 transgenic lines displayed the expected amplicons of the HptII gene fragments at 297 bp (Figure S3). Moreover, the transcription levels of the DaSOD1 gene were further monitored in six T₃ transgenic lines. Real-time qPCR analysis revealed that the expression of DaSOD1 gene was significantly higher in all transgenic lines compared to the WT plant (Figure 5). Among these transgenic lines, DaSOD-1 and DaSOD-3 exhibited the highest abundance of DaSOD1 transcript levels, suggesting that these two DaSOD1-overexpressing plants could be selected for Cd resistance assay.

Figure 5. Expression analysis of the DaSOD1 gene in transgenic tobacco plants overexpressing DaSOD1. Total RNA was extracted from the leaves of WT plant and DaSOD1-overexpressing lines (DaSOD1-6). Data are the means (n = 3) with corresponding standard deviations. Statistical significance among the WT and DaSOD1-overexpressing plants was determined by Student’s t-test (*P < 0.05; **P < 0.01).

Cadmium resistance in DaSOD1-overexpressing tobacco plants

DaSOD-1 and DaSOD-3 transgenic lines were selected to assess their resistance to Cd stress due to their high expression levels of the DaSOD1 gene (Figure 5). After 200 µM CdCl₂ treatment for 10 days, enhancements in Cd resistance were observed in the DaSOD-1 and DaSOD-3 transgenic lines (Figure 6a). The WT plants treated with CdCl₂ appeared yellow and eventually died, whereas the Cd-treated transgenic lines displayed green and healthy phenotypes. In consistency with the resistant phenotypes under CdCl₂ treatment, overexpression of DaSOD1 significantly alleviated the inhibition rate of root growth (Figure 6b). Additionally, the chlorophyll contents in the DaSOD1-overexpressing plants did not differ from those in the WT plants under non-stress conditions. However, following the CdCl2 treatments, the contents of both Chl a and Chl b in the DaSOD1-overexpressing plants were significantly increased (P < 0.05) compared to the WT plants (Figure 6c).

Figure 6. Cadmium tolerance assay of WT and DaSOD1-overexpressing plants. (a) Effect of cadmium treatment (200 μM CdCl₂) on the growth of WT and transgenic plants (DaSOD-1 and DaSOD-3). The representative photographs of WT and transgenic lines were taken after 10 days of cadmium exposure. (b) Effect of 200 μM CdCl₂ treatment on the inhibition rate of root length in WT and transgenic plants. (c) Effect of 200 μM CdCl₂ treatment on the chlorophyll a and chlorophyll b contents in WT and transgenic plants. Data are the means (n ≥ 5) with corresponding standard deviations. Statistical significance between the WT and transgenic plants was determined by Student’s t-test (*P < 0.05; **P < 0.01).

Cadmium toxicity adversely affects seed germination, ultimately undermining plant growth and productivity (Kaur et al. 2023). Thus, seed germination tests were performed under both non-stress and Cd-treated conditions to examine the resistance of transgenic seeds to Cd-induced stress. Under both non-stress and 100 µM CdCl₂ conditions, no significant differences in seed germination rates were observed between the transgenic and WT plants (Table 1). Nevertheless, when the plants were subjected to 200 µM CdCl₂, the germination rates of DaSOD-1 and DaSOD-3 seeds were significantly higher than those of WT seeds.

Table 1. The seed germination rates between the WT and transgenic plants under cadmium treatments.

Treatment	Plant	Seed germination rate (%)
		4 d	6 d	8 d
0 µM	WT	17.4 ± 2.5	86.9 ± 5.3	95.7 ± 1.5
	DaSOD-1	20.6 ± 3.4	90.5 ± 2.5	100 ± 0
	DaSOD-3	17.9 ± 2.4	84.1 ± 3.4	95.3 ± 1.3
100 µM	WT	3.5 ± 1.8	77.1 ± 18.1	91.2 ± 4.3
	DaSOD-1	29.3 ± 10.2*	86.9 ± 4.3	93.1 ± 1.2
	DaSOD-3	2.7 ± 1.8	85.1 ± 5.6	90.4 ± 4.2
200 µM	WT	4.3 ± 2.5	62.6 ± 5.4	72.6 ± 1.8
	DaSOD-1	20 ± 5.3*	75.1 ± 3.3*	82.5 ± 7.3*
	DaSOD-3	7.9 ± 3.2	82.7 ± 8.5*	92.2 ± 2.9*

Data (means ± SD) represent three independent replicates with at least 50 seeds counted per replicate.

Asterisk indicates a significant difference between WT and transgenic plants (*P < 0.05).

Elevation of Cd-induced oxidative stress tolerance in DaSOD1-overexpressing tobacco plants

The levels of superoxide radicals and MDA were used as indicators to monitor oxidative damage and lipid peroxidation in the DaSOD1-overexpressing lines and WT plant under CdCl₂ exposure for 7 days. The levels of superoxide radicals were elevated in both transgenic and WT plants following the CdCl₂ treatments, while the WT plant exhibited higher levels of superoxide radicals under the Cd treatments compared to the DaSOD-1 and DaSOD-3 plants, even under the non-stress conditions (Figure 7a). Differences in MDA levels were also examined between the transgenic and WT plants. The MDA levels in DaSOD-1 (5.50 nmol g⁻¹ FW) and DaSOD-3 (4.81 nmol g⁻¹ FW) plants were significantly lower than those in the WT plants (9.35 nmol g⁻¹ FW) after exposure of 100 µM CdCl₂. Similarly, when the plants were exposed to 200 µM CdCl₂, lower MDA levels were also detected in the two transgenic lines, whereas no difference in MDA levels was observed between the transgenic and WT plants under non-stress conditions (Figure 7b). Furthermore, cell viability in the roots of transgenic and WT plants was confirmed using trypan blue staining. The staining results revealed that more cells in the roots of WT plants, especially in the root tips, died due to CdCl₂ treatment. In contrast, fewer stained cells were observed in the roots of transgenic lines (Figure 7c). All the results indicated that the cell damages induced by Cd stress, including superoxide radical accumulation, lipid peroxidation, and cell injury, could be diminished by the presence of DaSOC1 in transgenic tobacco plants.

Figure 7. Cadmium-induced oxidative damage and cell death in the WT and transgenic plants. (a) Superoxide anion content of WT and transgenic plants under CdCl₂ treatments for 10 days. (b) MDA levels in the WT and transgenic plants under CdCl₂ treatments for 10 days. (c) Trypan blue staining of cadmium-induced cell injury in root tips of WT and transgenic plants under 200 μM CdCl₂ for 10 days. Data are the means (n ≥ 3) with corresponding standard deviations. Asterisk indicates a significant difference between WT and transgenic plants determined by Student’s t-test (*P < 0.05; **P < 0.01).

Discussion

High levels of heavy metal accumulation are known to induce ROS overproduction in plants, ultimately leading to accelerated cell death. Therefore, the inducible elevation of efficient peroxide detoxifications and enzyme protectors determines the defense ability of plants against heavy metal stress (Miller et al. 2008). The regulatory mechanisms of plants in response to Cd stress include antioxidant and metal chelation systems. However, several reports have suggested that antioxidant defense plays a more important role in Cd resistance of plants than phytochelatin (Wójcik et al. 2005; Sun et al. 2007; Sun et al. 2010). Although the antioxidant defense systems in response to heavy metal stress have been investigated in many plants, the correlation between antioxidant enzyme activation and heavy metal resistance remains complicated and varies significantly among different plant species (Meng et al. 2019). In this study, the differential responses of antioxidant enzyme activity were examined in the Cd-tolerant yam MH1 and -sensitive yam MH3. Among the four ROS-scavengers, only the SOD activity of MH1 exhibited a 2.12-fold increase at 14 days and a 2.49-fold increase at 28 days of CdCl₂ treatment compared to the mock treatment (Figure 2). In addition, the SOD activity in MH1 was significantly higher than that in MH3 under CdCl₂ treatment, while no difference in Cd accumulation was observed between the two cultivars. This suggests that the detoxification mechanism is crucial in MH1 in response to Cd stress. SOD functions as the central cellular defense against ROS damage by converting highly toxic superoxide radicals into less toxic hydrogen peroxide and molecular oxygen (Gupta et al. 1993). Thus far, the changes of SOD activity in response to Cd stress have been investigated in various plant species. In rice (Shah et al. 2001), red seaweed (Kumar et al. 2012), tomato (Alves et al. 2020), and alfalfa (Chen et al. 2022), the SOD activity was significantly increased following CdCl₂ treatment. However, not all plants increase their SOD activity when faced with Cd stress. The SOD activity of sugar cane (Fornazier et al. 2002), pea (Romero-Puertas et al. 2007), lettuce (Meng et al. 2019; Kolahi et al. 2020), sorghum (Jawad Hassan et al. 2020), and Sassafras tzumu (Zhao et al. 2021) was decreased or not altered significantly under CdCl₂ treatment. Moreover, Alves et al. (2020) reported that improvement of SOD activity is an efficient management strategy to reduce the negative impacts of Cd toxicity. Similarly, Meng et al. (2019) and Jawad Hassan et al. (2020) indicated that Cd-tolerant plant species and cultivars exhibited higher SOD activity under Cd stress. These findings highlight the importance of increasing plant SOD activity to cope with Cd stress, and provide an explanation for the observed Cd resistance in MH1 under Cd stress by increasing the SOD activity.

The activity of SOD in plants, including Cu/Zn-SOD, Mn-SOD, and Fe-SOD, is coordinated by the expression of the corresponding SOD genes under various conditions. (Huo et al. 2022). A complete functional identification of the key SOD gene may be helpful to enrich our understanding of Cd resistance in MH1 plants. Determining plant gene expressions at the transcriptional levels is a crucial step in identifying candidate genes (Chen et al. 2019; Kaur et al. 2023). In the present study, we cloned the DaSOD1 gene, and its gene expression pattern was closely correlated with the increased SOD activity under CdCl₂ treatments (Figure 3). Plant SOD exists in multiple isoforms that are found in distinct subcellular compartments and exhibits diverse properties when responding to environmental stress (Malecka et al. 2001). Based on sequence analysis and a phylogenetic tree, DaSOD1 belongs to the category of Cu/Zn-SOD (Figure 4 and Figure S2). In general, the Cu/Zn-SOD isoforms are widely distributed in cytoplasm, peroxisomes, and chloroplast (Stephenie et al. 2020), which aligns with the protein localization prediction of DaSOD1 in the cytoplasm. In addition, it is worth noting that another candidate gene, TR44896 (Cu/Zn-SOD), was also upregulated after 28 days of CdCl₂ treatment (Figure 3). It is worthwhile to conduct further functional analysis of this gene in the future. However, due to the limitations and complexity of the available genome information for yam plants, there might be additional undiscovered SOD genes that are upregulated in response to Cd stress in MH1.

Functional characterization of candidate genes cannot be conducted in many plants due to the limited availability of a transgenic system. Therefore, we ectopically expressed the DaSOD1 gene in tobacco to unveil the gene function. Overexpression of Cu/Zn-SOD genes can confer a certain level of resistance to various stresses in plants. For instance, transgenic Arabidopsis plants overexpressing Cu/Zn-SOD from Puccinellia tenuiflora exhibited improved resistance to multiple abiotic stresses (Wu et al. 2016). Overexpressing the Sedum alfredii Cu/Zn-SOD promoted seed germination and increased plant growth under Cd-induced oxidative stress in transgenic Arabidopsis (Li et al. 2017). Here, the effects of CdCl₂ treatment on the growth performance and physiological changes of transgenic tobacco plants overexpressing DaSOD1 were observed. Most WT plant exhibited marked chlorosis and growth impairment relative to the DaSOD1-overexpressing plants under CdCl₂ treatments (Figure 6a). The elevated chlorophyll levels in plants result in higher photosynthetic efficiency, supporting plant performance under adverse environments (Zhang et al. 2014; Chen et al. 2022; Li et al. 2022). Additionally, the rate of seed germination and root inhibition serves as another indicator of a plant’s resistance to Cd (Guan et al. 2009). Therefore, the overexpression of SOD1 alleviated the Cd-induced reduction in chlorophyll contents, root formation, and seed germination of tobacco plants (Figure 6b and c), supporting yam resistance to Cd stress. Cadmium-induced superoxide radicals cause toxicity, ultimately leading to cell death in plants (Garnier et al. 2006; Chen et al. 2022). DaSOD1 overexpression was involved in timely scavenging Cd-induced superoxide radicals, thereby reducing the MDA levels (Figure 7a and b). Obviously, the DaSOD1 gene plays a significant role in maintaining cell membrane integrality and contributes to enhanced cell viability in transgenic tobacco plants exposed to Cd stress (Figure 7c).

In conclusion, this study characterizes the DaSOD1 gene from the Cd-tolerant yam cultivar MH1. The elevation in the expression levels of the DaSOD1 gene play a crucial role in mediating stress-induced protective effects and detoxifying Cd-induced superoxide radicals in yam plants. This up-regulation of DaSOD1 gene may contribute to the increased superoxide dismutase activity observed in MH1 plants under Cd stress. In addition, some scientists have suggested that genetic modification of antioxidant enzymes, either by producing new isozymes or enhancing basal enzyme levels, can effectively mitigate stress-induced ROS accumulation in plant cells (Guan et al. 2009; Rajput et al. 2021). The transgenic tobacco plants were able to alleviate the excessive production of ROS induced by Cd, resulting in improved growth performance compared to the WT plants. As a result, the DaSOD1 gene holds substantial potential to serve as a candidate gene for plant bioengineering with the goal of producing highly resistant crop plants.

Author contributions

S-PC conceived and designed the research. SH, H-HL, QL, MW, ZH, S-PC performed the experiments. SH, H-HL, MW, QL, MW, ZH, and S-PC analyzed the data. S-PC wrote the manuscript. All authors have read and agreed to the final version of the manuscript.

Acknowledgements

We thank Lihong Li (Sanming Academy of Agricultural Sciences) for providing yam MH1 and MH3 seedling plants and Dr. Xuming Xu for experimental suggestion.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

This research was financially supported by the Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area. This study was also found by the Sanming City Science and Technology Bureau under grant 2021-N-13 to Zhehong Huang and the Chinese Culture University under grant PL11100884 to Shi-Peng Chen.

Notes on contributors

Shumei Hua

Shumei Hua is an associate research fellow at Institute of Dryland Crops, Sanming Academy of Agricultural Sciences.

Hsin-Hung Lin

Hsin-Hung Lin is an assistant professor at the Department of Agronomy, National Chung Hsing University.

Zhehong Huang

Zhehong Huang is an assistant research fellow at Institute of Dryland Crops, Sanming Academy of Agricultural Sciences.

Mengyao Wang

Mengyao Wang obtained her master degree in Agricultural Sciences, Fujian Agriculture and Forestry University.

Qing Li

Qing Li is an assistant research fellow at Institute of Dryland Crops, Sanming Academy of Agricultural Sciences.

Shi-Peng Chen

Shi-Peng Chen is an assistant professor at the Department of Horticulture and Biotechnology, Chinese Culture University. His research interests focus on the plant defense mechanisms in response to environmental stress and the interaction between plants and beneficial microorganisms.