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Zinc (Zn) is an essential trace element that plays a crucial role in plant growth and development, but excessive Zn can be stressful or even toxic to plants. The GLTP superfamily is critical for lipid metabolism and membrane stability maintenance, yet its function in plant Zn tolerance remains unclear. In this study, zinc stress treatment experiments were performed using transgenic apple calli, apple seedlings, Arabidopsis thaliana, and Solanum lycopersicum. Under Zn treatment, compared with the wild type (WT), the apple seedlings of the MbACD11 transgenic line exhibited significantly higher plant height and fresh weight, with increases of 5.87% and 93.21% respectively. Meanwhile, their MDA level, relative electrical conductivity, and accumulations of H2O2 and O2− were all significantly reduced, with decreases of 20.47%, 35.47%, 31.50%, and 36.78% respectively. Consistently, these data showed the same trend in calli, Arabidopsis thaliana, and tomato. These results demonstrated that the overexpression of MbACD11 significantly enhanced Zn tolerance in transgenic plants, and also verified that the function of this gene may be conserved across different species. In summary, this study establishes a molecular framework and theoretical basis for improving plant tolerance to Zn stress and paves the way for future mechanistic investigations.
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1. Introduction
Heavy metal pollution is getting worse in some parts of the world as a result of ongoing industrialization and technological improvement [1]. Tens of thousands of dangerous metals substances are released into the environment each year by industrial operations, according to statistics. After entering the soil, these hazardous industrial wastes not only contaminate the soil but also seriously endanger ecosystems [2]. Because of their stability, lack of microbial breakdown, and mineralization, heavy metals present a serious risk to human health and the ecosystem when they are present as contaminants in soils, especially agricultural soils [3].
One type of environmental contamination brought on by Zn and its components is called Zn pollution [4]. Mining, smelting, and metal processing are the main causes of Zn pollution, with the production of machinery being another significant factor. Additionally, emissions from sectors that greatly contribute to environmental pollution include papermaking, instrument manufacture, composite material manufacturing, and galvanization [5]. Plant physiological homeostasis is hampered by Zn pollution in agricultural soils, which is mostly caused by the overapplication of manure and the dumping of sludge. Chloroplast disintegration and oxidative membrane damage are two signs of phytotoxicity [6]. Due to dysregulated auxin polar transport, irrigation of wheat with wastewater containing Zn dramatically reduces germination efficiency, tiller development, and shoot elongation. Additionally, it severely deteriorates plant quality by preventing nutrients from being absorbed and allowing toxic compounds to build up, which eventually jeopardizes food safety [7]. Therefore, it is essential to design plants that accumulate Zn due to the combined requirements of ecotoxicology and crop physiology [8].
Plants adjust the expression of Zn transporters and chelators to maintain homeostasis in response to fluctuating soil Zn levels [9]. It has been genetically confirmed that the ZIP (ZRT-IRT-like) family is crucial for the distribution and uptake of Zn, especially in rice [10]. Those authors reported that glycolipid transfer protein (GLTP) domain proteins mediated the transit of glycerophospholipids, hence maintaining membrane homeostasis. Through phospholipid compositional changes, (Arabidopsis thaliana) AtGLTP controls plasma membrane Zn2+ permeability in Arabidopsis, suggesting new roles in Zn stress tolerance [11]. ACD11 is a specific sphingosine-1-phosphate (S1P) transporter that controls S1P flux, which in turn affects plant programmed cell death (PCD). Through S1P-induced NADPH oxidase activation, ACD11-dependent PCD restricts the spread of pathogens and produces reactive oxygen species (ROS) bursts that contain infection foci [12,13]. Mechanistic investigations showed that salicylic acid (SA)-induced ACD11-dependent PCD activation reduces infection [12]; plant PCD and abiotic stress adaptability are strongly correlated, according to recent studies [14].
Plants with elevated Zn levels experience growth retardation and leaf damage, which ultimately leads to death at lethal quantities [15,16]. Several species were used in a previous study to provide thorough phenotyping [17]. Apple calli and seedlings from MbACD11 transgenic lines have noticeably higher Zn tolerance. Through the coordinated downregulation of ROS-producing enzymes, the gene’s heterologous expression lowers oxidative stress and enhances Arabidopsis’s ability to adapt to Zn stress [18]. Cross-species functioning was confirmed by the tomato’s significantly increased Zn stress tolerance when MbACD11 was overexpressed [19]. This study clarifies the metal stress on ACD11 and offers theoretical frameworks for creating horticultural cultivars that are resistant to Zn. The aim of the present study was to explore a strategy for enhancing zinc stress resistance in plants.
2. Materials and Methods
2.1. Plant Materials and Growth Conditions
Calli from the ‘Orin’ apple (Malus domestica) were cultivated on Murashige and Skoog (MS) medium supplemented with 0.4 mg/L 6-benzyladenine (6-BA) and 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) [20]. Subculturing was carried out every 20 days while the cultures were kept at 25 °C in the dark [21].
Analysis was also done on apple seedlings, including wild-type Gala-3(GL-3) and ‘GL-3’-derived transgenic lines that overexpress MbACD11 plants [11]. For 28 days, the seedlings were grown on MS medium supplemented with 1 mg/L of indole-3-butyric acid (IBA) and 0.1 mg/L of 6-BA. Select a group of seedlings with consistent growth conditions. These apple seedlings were sliced into 1.0 cm-long stem pieces [22]. Following that, they were cultivated in medium MS with or without 700 µM ZnSO4. After 15 days of treatment, the phenotypes were seen and captured on camera, and the associated physiological indices were identified. Following rooting of apple seedlings on MS media, transgenic apple plants and wild-type apple seedlings were moved to an 8 × 8 cm2 nutrient bowl filled with nutrient soil and vermiculite (3:1, v:v) for two months in an adaptive growth chamber. Plants of uniform size were then grouped. The plants in the Zn treatment group and the control group had the same growth status. The course of treatment was 20 days long.
This study also used Arabidopsis thaliana, including three transgenic T3 lines that overexpress MbACD11 and the Columbia-0 (Col-0) wild-type. After being surface-sterilized and vernalized for three days at 4 °C, Arabidopsis seedlings were cultivated on 1/2 MS media with a 16-h light/8-h dark photoperiod, 22–24 °C, and 60% relative humidity [23]. Three-week-old Arabidopsis seedlings were then transplanted into soil supplemented with 700 µM of ZnSO4 for Zn stress treatment and grown for additional 14 days [24].
The study also included transgenic lines that ectopically express MbACD11 and wild-type tomatoes (‘Micro-Tom’). After 48 h of germination on damp filter paper, the seeds were moved to a growth chamber with white light (200 µmol photons m−2 s−1), a temperature regime of 25 °C during the day and 18 °C at night, and a light/dark cycle of 16/8 h. Following germination, seedlings were moved into soil that contained 700 µM ZnSO4, where they were raised for 14 days. Adjustments were made based on the laboratory’s previous research [25].
2.2. Genetic Transformation
Cotyledon explants were transformed using Agrobacterium tumefaciens strain EHA105 to produce the MbACD11-OE transgenic tomato. qRT-PCR was used to determine the levels of MbACD11 expression following kanamycin screening. All the primers that were used for the gene expression analysis are listed in Supplementary Table S1. This adjustment is based on previous research methodology [26]. All the primers used for gene expression analysis are listed in Supplementary Table S1.
2.3. RNA Extraction and Real-Time Quantitative PCR
RNA extraction: The Omini Plant RNA Kit (DNase I) (Cowin Biotech, Taizhou, China) was used to extract total RNA. Reverse transcription: The Prime Script First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) was used to create single-stranded cDNA from RNA. Real-time quantitative PCR: 18S rRNA served as the internal control, and Ultra SYBR Green Mixture (TakaRa) was used for amplification. Data analysis: The 2^(−ΔΔCT) technique was used to quantify relative gene expression. This test protocol was developed by reviewing relevant literature and adapting it to the present experimental setup in the lab [27].
2.4. Malondialdehyde (MDA) Content and Relative Electronic Conductivity Measurements
MDA assay: After homogenizing the samples in phosphate buffer in an ice bath, they were exposed to 5% thiobarbituric acid (TBA) for 20 min at 95–100 °C. MDA-TBA adduct generation was measured spectrophotometrically after cooling [28]. Relative electronic conductivity assay: A DDS-12 conductivity meter was used to weigh, pre-treat, and measure the relative electronic conductivity of equal amounts of experimental material [29].
2.5. Determination of Chlorophyll Content
95% ethanol (1:1, w/v) was used to soak fresh apple leaves. The extraction was carried out in total darkness to avoid light-induced chlorophyll degradation. Extraction of leaf samples was carried out using 95% ethanol for a 24-h period, after which the samples were subjected to spectrophotometric analysis at 649 nm and 665 nm [23].
2.6. Determination of Anthocyanin Content
Anthocyanins were extracted using the methanol-hydrochloric acid method. Plant material (0.05 g) was soaked in 5 mL of extraction solvent (85:15 v/v 95% methanol: 1.5 M HCl) for 24 h at room temperature in darkness [30]. The mixture was centrifuged at 5000 rpm for 5 min at 4 °C. The anthocyanin content of the extracted samples was measured at wavelengths of 530, 620 and 650 nm using a spectrophotometer [31,32].
2.7. Determination of Reactive Oxygen Species
The hydrogen peroxide (H2O2) content and superoxide anion (O2−) production rate were measured spectrophotometrically using commercial assay kits (Keming, Suzhou, China). Every test was carried out in accordance with the manufacturer’s instructions [33].
2.8. Statistical Analysis
Each experiment was performed with at least three biological replicates, and each replicate was set up with at least three technical replicates. Significant differences were analyzed for all data using the Data processing system (DPS v. 9.01) software. Different letters indicate significant differences (one-way ANOVA, Tukey–Kramer test, p < 0.05) [34].
3. Results
3.1. Expression Pattern of MbACD11 Under Zn Stress
Apple seedlings were treated with 700 μM of ZnSO4 in order to examine the possible involvement of MbACD11 in the response to Zn stress. The expression dynamics of MbACD11 were examined using qRT-PCR, and the results showed that MbACD11 transcript levels increased significantly to a peak after one hour of treatment, with an increase of 937.62% (Figure 1). These results suggest that MbACD11 contributes to apples’ early response to Zn stress.
3.2. MbACD11 Enhances the Resistance of Apple Calli to Zn Stress
To evaluate the biological function of MbACD11, three independent ‘Orin’ apple callus lines were compared with the WT [11].
To investigate the role of MbACD11 in Zn stress response, MbACD11-OE and WT apple calli were cultured on MS medium containing 700 μM ZnSO4 for 20 days (with normal MS medium as control). The experimental results show that there were no significant growth differences between the two under normal conditions. However, under Zn stress, MbACD11-OE exhibited significantly greater proliferation capacity than WT (Figure 2A). Specifically, the fresh weight of transgenic apple calli increased by 57.74% compared with that of WT (Figure 2B). Additionally, under Zn stress, the relative electronic conductivity and MDA content of MbACD11-OE decreased by 17.02% and 42.09%, respectively, compared with the WT (Figure 2C,D). These results indicate that MbACD11 overexpression enhances Zn stress resistance in apple calli.
3.3. MbACD11 Enhances Tolerance to Zn Stress in Apple Tissue Cultures
To further validate this hypothesis, this study investigated the role of the MbACD11 gene in zinc stress tolerance by comparing Zn-stressed transgenic apple seedlings overexpressing MbACD11 with WT plants. Under control conditions, both MbACD11-OE and WT plants grew well (Figure 3A). However, under zinc stress, both lines exhibited growth inhibition to varying degrees. Compared to WT plants, MbACD11-OE transgenic apple seedlings showed a higher resistance to Zn stress. In contrast to WT seedlings, which exhibited substantial anthocyanin accumulation under Zn stress, MbACD11-OE transgenic apple seedlings exhibited a minor buildup of anthocyanins while retaining their green hue. Under normal conditions, both WT and MbACD11-OE seedlings grew vigorously. After treatment with 700 µM of ZnSO4, the MbACD11-OE seedlings experienced less suppression in plant height compared to the WT (Figure 3B).
Next, this study examined physiological markers in both the Zn stress and control settings. There were not many differences between the two groups in the control group. However, during Zn stress, MbACD11-OE plants showed a 42.70% lower relative electronic conductivity, a 60.72% lower anthocyanin concentration, a 4.45% greater fresh weight, and a 139.56% greater chlorophyll content compared to the WT (Figure 3C–H). Thus, the present findings indicate that MbACD11 positively regulates Zn stress tolerance in plants.
3.4. Overexpression of MbACD11 Positively Regulates Zn Tolerance in Apple Rooted Seedlings
To assess the impact of MbACD11 overexpression on zinc tolerance, WT and MbACD11-OE apple rootstock seedlings were potted in soil and subjected to Zn stress. Under control conditions, no morphological or growth differences were observed between WT and transgenic seedlings.
However, under Zn stress, the growth of WT seedlings was significantly inhibited, whereas MbACD11-OE seedlings were less affected (Figure 4A,B). Consistent with this phenotype, Zn-stressed WT seedlings exhibited significantly lower fresh weight and plant height by 93.21% and 5.87%, respectively, compared to the transgenic lines (Figure 4C,D). Following Zn stress treatment, WT seedlings exhibited increased relative electrical conductivity and MDA content by 35.47% and 20.47%, respectively, compared to MbACD11-OE seedlings (Figure 4E,F). The greater sensitivity of WT seedlings to Zn stress was indicated by their significantly higher levels of H2O2 and an increased production rate of O2− by 31.50% and 36.78%, respectively, relative to the MbACD11-OE line (Figure 4G,H). These results demonstrate that overexpression of MbACD11 significantly enhances Zn stress tolerance in apple rootstock seedlings.
3.5. MbACD11 Enhances Zn Stress Tolerance in Arabidopsis Seedlings
For this investigation, this study used three Arabidopsis lines heterologously expressing MbACD11, as previously described [11].
The phenotypic differences between WT and transgenic MbACD11-OE Arabidopsis seedlings treated with 700 μM ZnSO4 were evaluated in order to examine the role of MbACD11 in response to Zn stress. There was no discernible difference in the growth of transgenic MbACD11-OE Arabidopsis seedlings and WT seedlings under normal growth conditions. In contrast to WT, MbACD11-OE plants had improved resistance to Zn stress, as evidenced by 22.01%, 38.10%, and 93.33% increases in primary root length, fresh weight, and lateral root number, respectively (Figure 5A–D). The transgenic plants also exhibited a markedly lower relative electrolyte conductivity, with a decrease of 9.92% (Figure 5E). These findings suggest that by encouraging root growth, MbACD11 may improve plant resistance to Zn stress.
3.6. Ectopic Expression of MbACD11 in Arabidopsis Positively Regulates Zn Tolerance Through Enhanced Scavenging of ROS
A Zn-stressed atmosphere containing 700 μM of ZnSO4 was used to incubate Arabidopsis seedlings that were 3 weeks old. After treatment, this study discovered that there was no discernible difference between the transgenic Arabidopsis’s physiological indices and those of WT under control. However, when exposed to Zn, the transgenic Arabidopsis showed much higher fresh weight and plant height than the WT by 65.37% and 30.19%, respectively (Figure 6B,C). Under Zn stress treatment, the transgenic plants exhibited a 24.74% lower relative electrical conductivity and a 44.91% lower MDA level than the WT (Figure 6D,E). Through a variety of mechanisms, heavy metals infiltrate soil and plant environments, where they cause plants to create more ROS, which damage cell membranes and deactivate antioxidant enzymes, thus compromising the survival and function of plant cells. Thus, this study looked at whether MbACD11 might control ROS levels to withstand Zn ion stress. Following Zn treatment, measurements were made of the O2− and H2O2 levels in both WT and transgenic MbACD11-OE Arabidopsis. Transgenic MbACD11-OE plants and WT plants cultivated under normal circumstances did not vary significantly. However, under Zn stress, the leaves of transgenic MbACD11-OE plants accumulated significantly lower levels of O2− and H2O2 by 11.78% and 25.15%, respectively, than those of WT (Figure 6F,G). MbACD11 decreases oxidative damage and increases resistance to Zn stress. These findings imply that MbACD11 controls the generation of ROS to improve resistance to Zn stress. By lessening oxidative damage brought on by the buildup of ROS, it postpones the planned death of plant cells. Under Zn stress, this molecular pathway improves Arabidopsis survival.
3.7. MbACD11 Enhances Zn Stress Tolerance in Tomato Seedlings
This study also looked at how transgenic MbACD11 tomato plants responded to Zn stress because the MbACD11 gene in Arabidopsis positively influences Zn tolerance. This work created three distinct transgenic tomato lines with heterologous MbACD11 overexpression in order to examine the function of MbACD11 in plant Zn stress response. The study examined the phenotypic variations between transgenic MbACD11-OE tomato seedlings treated with 700 μM of ZnSO4 and Micro-Tom (WT) tomato seedlings (Figure 7A). According to the findings, the transgenic plants showed substantial Zn tolerance in comparison with the WT, as evidenced by 81.95% and 112.50% increases in fresh weight and root length, respectively (Figure 7B,C), and a 19.75% reduction in relative electrical conductivity following Zn stress treatment (Figure 7D). Interestingly, under typical growing conditions, there was no discernible difference between the two plant species. These findings support the idea that MbACD11 has a conserved regulatory mechanism for Zn tolerance across several plant species and corroborate its positive regulatory role in response to Zn stress.
Both wild-type and transgenic MbACD11-OE Micro-Tom tomatoes were grown as normal. Under controlled conditions, there was no difference between the wild-type and transgenic tomatoes. After three or four Zn stress treatments, transgenic MbACD11-OE tomatoes collected much more biomass than WT under typical growth circumstances (Figure 8A). Following Zn treatment, the transgenic MbACD11-OE tomatoes outpaced the WT in terms of fresh weight and height by 13.81% and 16.44%, respectively (Figure 8B,C). Furthermore, during Zn stress, the transgenic MbACD11-OE tomatoes had 66.23% lower MDA levels and 13.06% reduced relative electrical conductivity, respectively, in comparison with the WT (Figure 8 D,E). Compared to the WT, transgenic MbACD11-OE tomatoes had significantly reduced levels of O2− and H2O2 in their leaves by 36.11% and 31.26%, respectively (Figure 8F,G). Furthermore, the present results suggest that MbACD11 may enhance tolerance to Zn stress by reducing oxidative damage. These results demonstrate that MbACD11 overexpression significantly increases tomato Zn stress tolerance.
4. Discussion
The constantly developing global environment frequently results in circumstances that are detrimental to the growth and development of plants [35]. These alterations include abiotic stressors including drought, excessive soil heavy metals, cold, heat, salt, and nutritional shortages, as well as biotic stresses like bacterial infections and herbivory [36]. Environmental factors such as heavy metal stress can limit the range of natural plants, reduce agricultural output, and endanger food security [37]. One of the most serious abiotic risks to plant growth, development, quality, and production is heavy metal stress [38].
This study looked at how apple calli, apple seedlings, Arabidopsis, and tomato responded to Zn stress. Under Zn stress circumstances, apple calli, apple seedlings, Arabidopsis, and tomato overexpressing MbACD11 showed improved growth performance, higher height, and larger fresh weight in comparison to WT (Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8). These results indicate that overexpression of MbACD11 enhances plant resistance to zinc stress. As the primary organ for plant nutrient absorption, the root system serves as the first line of defense against heavy metals in the soil. Consequently, root toxicity symptoms are more pronounced than in other plant parts [39]. One of the earliest responses to high Zn stress is the inhibition of root growth, including reduced lateral root formation. Subsequently, roots may exhibit hardening, thickening, and impaired cell division and elongation at the root tip [14]. This study examined the responses of Arabidopsis and tomato to Zn stress. Under Zn stress, Arabidopsis and tomato plants overexpressing the MbACD11 gene displayed longer root lengths and more lateral roots than WT seedlings (Figure 5 and Figure 7). These results indicate that MbACD11-overexpressing plants may enhance their resistance to zinc stress by increasing root length and lateral root number [40]. Among the indicators of stress-induced damage, membrane lipid peroxidation is commonly assessed by measuring MDA levels [41]. MDA content reflects the extent of cell membrane damage [42]. Environmental stresses can induce oxidative damage to plant lipids, and MDA serves as a widely used marker for such damage. Several studies have examined MDA accumulation under various stress conditions in plants [43]. In contrast to WT controls, this study found that apple calli, apple seedlings, Arabidopsis and tomato that overexpressed the MbACD11 gene showed noticeably reduced levels of relative electrical conductivity and MDA under Zn stress conditions (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8). An increase in MDA content indicates elevated lipid peroxidation and a loss of membrane stability. These are both key indicators of oxidative stress under zinc conditions [44]. Many plant species experience electrolyte leakage, which can be brought on by abiotic stressors such as exposure to heavy metals, excessive salinity, and acidity of the soil. It reflects stress-induced cellular damage and is a useful indicator for assessing plant stress resistance [45]. Under stressful conditions such as drought, low temperatures, high salt levels, exposure to heavy metals and redox imbalances often occur in plants [46,47]. These imbalances trigger the excessive accumulation of ROS. Heavy metal stress triggers homeostatic regulatory mechanisms in plants to balance metal ions and regulate ROS signalling pathways. Studies have shown that ROS generation and signalling play an important role in the plant’s response to and tolerance of heavy metal toxicity. For example, excess Zn interferes with the dynamic balance of reactive oxygen species, such as H2O2 and O2−, leading to elevated ROS levels. In order to maintain normal physiological metabolism, plants depend on antioxidant systems to scavenge excess ROS and thereby reduce oxidative damage [48]. In line with earlier findings, this study discovered that, when exposed to Zn stress, the H2O2 and O2− levels of apple seedlings, Arabidopsis, and tomato overexpressing the MbACD11 gene were considerably lower than those of the WT control group (Figure 4, Figure 6 and Figure 8). The contents of H2O2 and O2− in calli, apples, Arabidopsis and tomatoes were measured. The results showed that MbACD11-OE plants exhibited lower levels of O2− and H2O2 under zinc stress conditions compared to WT plants, demonstrating enhanced tolerance to zinc stress [48]. This study reveals the positive regulatory role of the MbACD11 gene in plant zinc stress responses through multi-species validation. Furthermore, the mechanism by which it acts is closely associated with enhancing antioxidant capacity, maintaining cell membrane integrity and adapting to stressful environments by optimising root architecture. This provides important candidate genes and a theoretical basis for breeding crop varieties that can tolerate zinc stress through genetic engineering.
5. Conclusions
The results demonstrated that the expression of MbACD11 induced specific phenotypic changes in transgenic plants, effectively reducing ROS accumulation and enhancing cell membrane stability. As a result, this dual impact greatly increased the ability of several plant species, such as Arabidopsis thaliana, tomato, and apple, to withstand Zn stress. Our research increases knowledge of MbACD11’s stress-reduction processes and offers perspectives on the biological function of the protein in apples. However, the mechanism by which MbACD11 enhances zinc resistance by reducing reactive oxygen species (ROS) is unclear, providing a focus for future research.
Methodology, A.-S.W., D.-R.W. and X.L.; software, G.-L.C., Q.W., W.-L.J. and T.-T.W.; validation, A.-S.W., D.-R.W. and C.-L.Z.; investigation, C.-X.Y., A.-S.W. and D.-R.W.; data curation, Y.-Y.G., C.-H.C. and Y.-J.C.; writing—original draft preparation, A.-S.W.; writing—review and editing, D.-R.W., C.-L.Z. and C.-X.Y.; supervision, C.-X.Y. and C.-L.Z.; funding acquisition, C.-X.Y. and C.-L.Z. All authors have read and agreed to the published version of the manuscript.
The original contributions presented in this study are included in the article/
The authors declare no conflicts of interest.
The following abbreviations are used in this manuscript:
| ZIP | ZRT-IRT-like |
| GLTP | Glycolipid transfer protein |
| TLA | Sphingosine-1-phosphate |
| PCD | Programmed cell death |
| ROS | Reactive oxygen species |
| SA | Salicylic acid |
| MS | Murashige and Skoog |
| 6-BA | 6-benzyladenine |
| 2,4-D | 2,4-dichlorophenoxyacetic acid |
| IBA | Indole-3-butyric acid |
| MDA | Malondialdehyde |
| TBA | Thiobarbituric acid |
| H2O2 | Hydrogen peroxide |
| O2− | Superoxide anion |
Footnotes
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Figure 1 Expression patterns of MbACD11. The expression level of MbACD11 was determined under conditions with or without 700 μM ZnSO4 treatments for 0, 1, 3, 6, 12, and 24h, respectively. Data are mean ± SD of three independent replicates. Different letters represent significant differences (one-way ANOVA, p < 0.05).
Figure 2 MbACD11-OE exhibit enhanced resilience to Zn stress in apple calli. (A) The phenotypes of WT and MbACD11-OE apple calli with 700 μM ZnSO4 treatments. Bar = 1 cm. (B) Fresh weight, (C) relative electrical conductivity, and (D) MDA content of WT and MbACD11-OE transgenic apple calli. After analysis, the mean ± standard deviation (n = 3) was used to express the data. Lowercase letters (p < 0.05) denote significant differences within groups.
Figure 3 MbACD11-OE transgenic apple seedlings exhibit Zn resistance. (A,B) The phenotypes of WT and MbACD11-OE apple seedlings treated with or without 700 μM ZnSO4. Following Zn treatment, the following variables are shown: (C,D) anthocyanin content; (E) fresh weight; (F,G) chlorophyll content; and (H) relative electrical conductivity of WT and MbACD11-OE transgenic apple seedlings. Three independently conducted biological replicates were used to compute the standard deviation of the means (±SD, n = 3), which is shown by the error bars in this figure. Different letters indicate significant differences (p < 0.05). Bar = 1 cm.
Figure 4 MbACD11-OE transgenic apple-rooted seedlings exhibit Zn resistance. (A,B) The phenotypes of WT and MbACD11-OE apple-rooted seedlings treated with or without 700 μM of ZnSO4. (C) Plant height; (D) fresh weight of plant aboveground; (E) relative electronic conductivity; (F) MDA content; (G) the content of H2O2 and (H) production rate of O2− in WT and MbACD11-OE transgenic lines after Zinc treatment. Three independently conducted biological replicates were used to compute the standard deviation of the means (±SD, n = 3), which is shown by the error bars in this figure. Different letters (p < 0.05) indicate significant differences. Bar = 1 cm.
Figure 5 MbACD11 enhances the resistance of Arabidopsis seedlings to Zn stress. (A) Growth phenotypes of WT and transgenic Arabidopsis treated with nothing added or supplemented with 700 μM of ZnSO4; (B) root length; (C) fresh weight; (D) the number of lateral roots; and (E) the relative electronic conductivity. After data analysis, the mean ± standard deviation (n = 3) was used. Lowercase letters (p < 0.05) denote significant differences within groups. Bar = 1 cm.
Figure 6 MbACD11 enhances the resistance of Arabidopsis to Zn stress. (A) The phenotypes of WT and MbACD11-OE Arabidopsis under 700 μM ZnSO4 treatment and without exogenous ZnSO4 supplementation; (B) plant height; (C) fresh weight; (D) relative electronic conductivity; (E) MDA content; (F) H2O2 content; (G) O2− generation rates of WT and MbACD11-OE transgenic Arabidopsis under control and 700 μM ZnSO4 treatments. In this figure, error bars show the standard deviation of the means (±SD, n = 3), which was determined using three separate biological replicates. Different letters denote significant differences (p < 0.05). Bar = 1 cm.
Figure 7 MbACD11 enhances the resistance of tomato seedlings to Zn stress. (A) Phenotypes of WT and MbACD11-OE tomato seedlings with 700 μM of ZnSO4. (B) Analysis of root length in WT and MbACD11-OE transgenic tomato under 700 μM ZnSO4 treatment. (C) Analysis of fresh weight in WT and MbACD11-OE transgenic tomato under 700 μM ZnSO4 treatment. (D) Analysis of the relative electronic conductivity in WT and MbACD11-OE transgenic tomato under 700 μM ZnSO4 treatment. After analysis, the mean ± standard deviation (n = 3) was used to express the data. Lowercase letters denote significant differences within groups (p < 0.05). Bar = 1 cm.
Figure 8 MbACD11 enhances the resistance of tomato to Zn stress. (A) Phenotype of zinc tolerance in the tomato with ectopic overexpression of MbACD11; (B) plant height; (C) fresh weight; (D) MDA content; (E) relative electronic conductivity; (F) H2O2 content; and (G) O2− generation rates of WT and MbACD11-OE transgenic tomatoes under control and 700 μM ZnSO4 treatments. Data were analyzed and expressed as the mean ± standard deviation (n = 3). Significant differences within groups are indicated by lowercase letters (p < 0.05). Bar = 1 cm.
Supplementary Materials
The following supporting information can be downloaded at:
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