Ectopic Expression of AetPGL from Aegilops tauschii Enhances Cadmium Tolerance and Accumulation Capacity in Arabidopsis thaliana

Cadmium (Cd) is a toxic heavy metal that accumulates in plants, negatively affecting their physiological processes, growth, and development, and poses a threat to human health through the food chain. 6-phosphogluconolactonase (PGL) is a key enzyme in the Oxidative Pentose Phosphate Pathway(OPPP) in plant cells, essential for cellular metabolism. The OPPP pathway provides energy and raw materials for organisms and is involved in antioxidant reactions, lipid metabolism, and DNA synthesis. This study describes the Cd responsive gene AetPGL from Aegilops tauschii. Overexpression of AetPGL under Cd stress increased main root length and germination rate in Arabidopsis. Transgenic lines showed higher antioxidant enzyme activities and lower malondialdehyde (MDA) content compared to the wild type. The transgenic Arabidopsis accumulated more Cd in the aboveground part but not in the underground part. Expression levels of AtHMA3, AtNRAMP5, and AtZIP1 in the roots of transgenic plants increased under Cd stress, suggesting AetPGL may enhance Cd transport from root to shoot. Transcriptome analysis revealed enrichment of differentially expressed genes (DEGs) in the plant hormone signal transduction pathway in AetPGL-overexpressing plants. Brassinosteroids (BR), Gibbenellin acid (GA), and Jasmonic acid (JA) contents significantly increased after Cd treatment. These results indicate that AetPGL may enhance Arabidopsis’ tolerance to Cd by modulating plant hormone content. In conclusion, AetPGL plays a critical role in improving cadmium tolerance and accumulation and mitigating oxidative stress by regulating plant hormones, providing insights into the molecular mechanisms of plant Cd tolerance.


Introduction
As industry and agriculture continue to advance rapidly, the escalating issue of environmental heavy metals (HMs) pollution is a matter of increasing concern.Soil microorganisms are unable to break down toxic heavy metals such as cadmium (Cd), lead, mercury, and aluminum [1].Nevertheless, the deleterious effects of these heavy metals on plants are substantial, with even minute quantities exerting a detrimental influence on plant growth [2].Metal poisoning results in an imbalance in plant nutrition, sluggish growth, and alterations in essential physiological processes such as photosynthesis, respiration, and transpiration, ultimately culminating in the demise of the plant [3,4].These toxic heavy metals accumulate in plants and enter the food chain, posing a threat to the health Plants 2024, 13, 2370 2 of 19 of animals and humans.Excessive metal buildup in the human body has been linked to issues such as cancer and damage to organs such as the liver, kidneys, spleen, bones, and reproductive system, according to studies [5,6].As a non-essential element for plants, animals, and humans, Cd concentrations were reported to surpass the national standard by 7.0%, as per a government report in China [7].Consequently, elucidating the molecular mechanism of plant Cd tolerance and cultivating crops with robust Cd tolerance and minimal Cd accumulation have become pressing issues.
The oxidative pentose phosphate pathway (OPPP) is a classical metabolic route in plants [8].Key enzymes within this pathway, including 6-phosphogluconolactonase (PGL), 6-phosphogluconate dehydrogenase (6PGD), and glucose-6-phosphate dehydrogenase (G6PD), are essential for NADPH (Nicotinamide Adenine Dinucleotide Phosphate) production.NADPH acts as a critical reducing agent, aiding in the elimination of reactive oxygen species (ROS) [9,10].Thus, the OPPP is crucial for regulating plant growth, development, and responses to various environmental stresses.Increased Cyt-G6PDH activity enhances drought resistance in soybean roots through ABA-dependent signaling [11,12].Transgenic tobacco plants overexpressing G6PDH show greater cold tolerance than controls, as well as increased activities of superoxide dismutase (SOD) and peroxidase (POD), improving freezing resistance in poplar [13].Additionally, G6PDH modulates cellular redox balance and oxidative stress responses by affecting H + -ATPase and Na + /H + antiporters in the plasma membrane and participating in antioxidant synthesis under salt and drought conditions [14,15].The expression and activity of TaG6PD and Ta6PGD are upregulated in winter wheat under cold stress, a response further enhanced by exogenous abscisic acid (ABA).When overexpressed in Arabidopsis, TaG6PD and Ta6PGD increase ROS-scavenging ability and survival rates compared to wild-type plants under cold stress [16].Similarly, the transcript levels of Os6PGDH1 and Os6PGDH2 rise in rice seedlings exposed to drought, cold, high salinity, and ABA treatments [17,18].Studies have shown that Gm6PGDH1 overexpression enhances soybean tolerance to phosphate starvation by improving root development and antioxidant system modulation [19].Unlike G6PD and 6PGD, the role of PGL in plant responses to abiotic stress has been less studied.Recent findings emphasize PGL's significant role in bacterial pathogen resistance [20] and regulation of the glucose metabolism pathway [21,22].
Plant hormones are increasingly recognized by researchers as an eco-friendly method to enhance plant tolerance to HM stress.These hormones, produced in minute quantities, act as chemical messengers, crucially regulating plant growth and development.Additionally, they enhance plant resistance to various stresses [23].Early studies have shown that exogenous application of plant hormones can improve plant tolerance to HM exposure [24].For example, foliar application of 0.1 mM salicylic acid (SA) significantly improved the physiological characteristics of rice seedlings under Cd stress by reducing the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) [25].Similarly, applying auxin (IAA) to mustard plants mitigated As-induced oxidative damage by decreasing reactive oxygen species (ROS) and lipid peroxidation while enhancing membrane stability and rigidity [26].Furthermore, HM exposure causes significant changes in endogenous plant hormone levels, regulating various plant stress adaptation mechanisms [27].Experiments on Lycium chinense under Cd stress revealed the protective role of ethylene, showing that upregulation of the LchERF gene and accumulation of oxidized glutathione (GSG) significantly increased endogenous ethylene production, thereby enhancing Cd stress tolerance [28].Moreover, HMA4 (Heavy Metal ATPase 4) is responsible for transporting Cd from the roots to the stem and accumulating it in stem tissue.Upregulation of HMA4 can also modify endogenous ABA levels, reducing oxidative damage and toxicity caused by Cd in plants [29].
Common wheat (Triticum aestivum L.) ranks among the world's most extensively cultivated crops, featuring three genomes (A, B, and D).Aegilops tauschii, the D genome donor of common wheat, exhibits abundant genetic diversity and resilience to various biotic and abiotic stresses [30,31].In a previous study, differentially expressed genes (DEGs) under Cd stress were discerned in Ae. tauschii through transcriptome sequencing.Additionally, our investigation revealed significant upregulation of the AetPGL gene under Cd stress.This study predominantly elucidates the role of AetPGL, overexpressed in A. thaliana, and demonstrates its capacity to enhance Cd tolerance and accumulation in transgenic plants by augmenting phytohormone synthesis.The findings deepened the understanding of the mechanism of AetPGL's functions as a key component in the phytohormone-mediated signaling pathway in Cd stress.

Plant Culture and Treatments
The experimental plant materials comprised Ae. tauschii and A. thaliana.A. thaliana seeds were disinfected using 75% ethanol for 3 min, followed by rinsing with sterile water and 2% NaClO for 10 min each, and finally rinsed with sterile water three times to remove any residual NaClO on the seed surface.After washing, the seeds underwent vernalization at 4 • C for 3 days, germinated vertically on Murashige and Skoog (MS) medium for 7 days, and were subsequently transplanted into nutrient soil.After three weeks of growth in nutrient soil, nine pots of wild-type plants and three pots of transgenic lines (each pot containing four plants) were selected and divided into three sets.The plants were cultivated in Hoagland nutrient solution for 14 days, with an option to include or exclude 2.5 mmol/L CdCl 2 .A. thaliana seeds disinfected as described above were placed in 1/2 MS medium containing 100 µM and 150 µM Cd, followed by vernalization at 4 • C for three days.Observations on root length and germination rate were made after 11 days of growth under standard conditions.

Phylogenetic and Conserved Motif Analysis of AetPGL Proteins
We used the PGL protein sequence as the query condition to search the protein sequence in the NCBI database.The AetPGL sequence was aligned with related proteins using DNAMAN 5.2.2.Subsequently, phylogenetic relationships were analyzed employing the maximum likelihood method and visualized using MEGA 7.0.The genetic phylogenetic tree for the 15 PGL proteins was constructed using the maximum likelihood (ML) method (model: Jones-Taylor-Thornton).A total of 1000 bootstrap replicates were performed using MEGA 7.0.

RNA Isolation and qRT-PCR
RNA extraction and RT-qPCR were conducted in accordance with the procedures outlined in the published research [32].The RT-qPCR was performed using the BIOER FQD-48A system (BIOER, Hangzhou, China).The primer sequences used in this analysis are available in the attached Table, with actin 1 utilized as the reference gene.The 2 −∆∆CT method, which was reported previously, was employed to estimate gene expression [33].

Construction of the AetPGL Expression Vector and Genetic Transformation of Arabidopsis thaliana
The RNA extracted from Ae. tauschii was reverse transcribed into cDNA using the ExonScript RT Mix (Bgbiotech, Chongqing, China).The CDS sequence of AetPGL was amplified with a high-fidelity enzyme (Yeasen, Shanghai, China), using primers listed in Table A1, and ligated into the pBI121 plasmid using the Hieff Clone ® Plus One Step Cloning Kit (Yeasen, Shanghai, China) to form the recombinant plasmid pBI121-AetPGL [34].The recombinant plasmid pBI121-AetPGL was then transformed into Agrobacterium tumefaciens strain GV3101.After the wild-type A. thaliana began flowering, genetic transformation was carried out using the floral dip method to introduce the recombinant Agrobacterium GV3101 carrying the target gene into the wild-type A. thaliana [35].Positive plants were selected on 1/2 MS medium containing 50 mg/L kanamycin.T3 generation seeds were obtained through multiple rounds of screening and used for subsequent experiments.

Phenotypic Analysis and Physiological Index Determination
The activities of antioxidant enzymes (POD; SOD; peroxidase, APX; catalase, CAT), along with the concentrations of malondialdehyde (MDA) and proline (Pro), were measured and analyzed in both the shoots and roots of WT and transgenic Arabidopsis.Quantification was conducted using a test kit with detailed experimental procedures (Solarbio, Beijing, China).Additionally, the concentrations of cytokinin (CK), brassinosteroids (BR), gibberellic acid (GA), and jasmonic acid (JA) in the aerial parts of both wild-type and transgenic Arabidopsis were assessed.Quantification was performed using a test kit with detailed experimental procedures (Jingmei Biological Technology, Yancheng China).

Determination of Cadmium Content
Cd accumulation in the roots and shoots of both the OE2 and the WT was determined.It is crucial to note that the plants should be thoroughly washed to eliminate any external Cd that may impact the determination results.The cleaned and collected plant samples are then subjected to a 7-day drying period in an oven to ensure the complete removal of any moisture.Subsequently, the thoroughly dried plants are finely ground into a powder.Finally, the content measurements are conducted using ICP-MS, following the specific procedural steps outlined in the published paper [36].

RNA-Seq Analysis
We subjected both the WT and transgenic Arabidopsis to treatments with 0 mM CdCl 2 and 2.5 mM CdCl 2 .Subsequently, the aboveground portions were selected for transcriptome sequencing.Then, RNA was extracted from plant tissues using the EASY spin Plant RNA extraction kit (Aidlab, Beijing, China).The purity of the extracted RNA, as determined by OD260/280 and OD260/230 ratios, was assessed using the Nanodrop (2000) spectrophotometer (Thermo Scientific, Waltham, MA, USA).Following this, RNA integrity and the presence of DNA contamination were evaluated through 1.5% agarose gel electrophoresis (PAGE).To minimize the potential impacts of RNA structural variations on sequencing results, the Agilent 2100 biological analyzer software (Santa Clara, CA, USA) was utilized for precise RNA integrity assessment.Following library construction, initial quantification was conducted using Qubit 3.0 software.The Illumina NovaSeq 6000 sequencer (San Diego, CA, USA), renowned for its advanced sequencing technology, was then employed for transcriptome sequencing after a comprehensive library inspection.Real-time quantitative PCR (RT-qPCR) was subsequently employed to validate the accuracy of the transcriptome data.

Statistical Analysis
Regression analysis was conducted using SPSS V25, which is appropriate for evaluating variance among groups.Each treatment group and the data presented in the article were subjected to the experiment three times.

Primers
All the primers used in this study are listed in Table A1.

AetPGL Conserved Motif and Phylogenetic Analysis
The PGL protein is conserved among these 15 proteins, and sequence alignment reveals that AetPGL shares the same protein domain as PGL in other plants (Figure 1a).To gain a deeper insight into the phylogenetic relationships among these 15 proteins, a phylogenetic analysis of protein sequences was conducted using MEGA7.0software.The evolutionary analysis indicates that the PGL protein of Ae. tauschii is most closely related to the PGL protein in wheat (Figure 1b).evolutionary analysis indicates that the PGL protein of Ae. tauschii is most closely related to the PGL protein in wheat (Figure 1b).

Cd-Induced AetPGL Expression
RT-qPCR results showed that under normal conditions (0 mM Cd), AetPGL was not expressed in roots and shoots.However, after applying 2.5 mM Cd stress, significant expression occurred in roots and shoots, mRNA levels were significantly increased, and the mRNA level in shoots was higher than that in roots.The results showed that AetPGL was a key gene in response to Cd stress, and its expression was upregulated under Cd stress (Figure 2a).

Cd-Induced AetPGL Expression
RT-qPCR results showed that under normal conditions (0 mM Cd), AetPGL was not expressed in roots and shoots.However, after applying 2.5 mM Cd stress, significant expression occurred in roots and shoots, mRNA levels were significantly increased, and the mRNA level in shoots was higher than that in roots.The results showed that AetPGL was a key gene in response to Cd stress, and its expression was upregulated under Cd stress (Figure 2a).evolutionary analysis indicates that the PGL protein of Ae. tauschii is most closely related to the PGL protein in wheat (Figure 1b).

Cd-Induced AetPGL Expression
RT-qPCR results showed that under normal conditions (0 mM Cd), AetPGL was not expressed in roots and shoots.However, after applying 2.5 mM Cd stress, significant expression occurred in roots and shoots, mRNA levels were significantly increased, and the mRNA level in shoots was higher than that in roots.The results showed that AetPGL was a key gene in response to Cd stress, and its expression was upregulated under Cd stress (Figure 2a).

Overexpression of AetPGL Enhanced the Tolerance of Arabidopsis to Cd
We generated 11 transgenic lines using the inflorescence infection method.The expression levels of these 11 lines were assessed through fluorescence quantitative RT-qPCR, and three overexpression lines (OE2, OE4, and OE6) exhibiting the highest expression levels were chosen for subsequent experiments (Figure 2b).Under normal medium conditions (1/2 MS), there were no significant differences in root length and germination rate between the WT and overexpression lines.However, in 1/2 MS medium containing 100 µM and 150 µM Cd, the root growth and seed germination of the WT were severely inhibited.The roots were significantly shorter compared to the transgenic lines, and the germination rate was markedly lower (Figure 3a,b).Consequently, we quantified the root length and germination rate (Figure 4a,b).Under normal conditions, the growth patterns of both WT and transgenic lines remained consistent.However, after 7 days of treatment with 2.5 mM Cd, the growth of the WT was inhibited, and the leaves exhibited a significantly higher degree of yellowing compared to the overexpression lines.This suggests that overexpression of AetPGL enhances Cd tolerance in transgenic Arabidopsis (Figure 3c).
We generated 11 transgenic lines using the inflorescence infection method.The expression levels of these 11 lines were assessed through fluorescence quantitative RT-qPCR, and three overexpression lines (OE2, OE4, and OE6) exhibiting the highest expression levels were chosen for subsequent experiments (Figure 2b).Under normal medium conditions (1/2 MS), there were no significant differences in root length and germination rate between the WT and overexpression lines.However, in 1/2 MS medium containing 100 µM and 150 µM Cd, the root growth and seed germination of the WT were severely inhibited.The roots were significantly shorter compared to the transgenic lines, and the germination rate was markedly lower (Figure 3a, b).Consequently, we quantified the root length and germination rate (Figure 4a, b).Under normal conditions, the growth patterns of both WT and transgenic lines remained consistent.However, after 7 days of treatment with 2.5 mM Cd, the growth of the WT was inhibited, and the leaves exhibited a significantly higher degree of yellowing compared to the overexpression lines.This suggests that overexpression of AetPGL enhances Cd tolerance in transgenic Arabidopsis (Figure 3c).

Overexpression of AetPGL Enhanced the Antioxidant Capacity of Transgenic Arabidopsis
We measured the physiological indices of the roots and shoots in both the WT and overexpression lines.The contents of Pro and MDA can serve as indicators to validate plant damage under stressful conditions.In this study, following Cd stress, the MDA content in the WT significantly exceeded that of the transgenic lines (Figures 5a and 6a).Conversely, the Pro content was noticeably lower in the WT compared to the transgenic lines (Figures 5b and 6b), suggesting more severe damage to the cells of WT plants.Furthermore, Cd stress increased the activities of antioxidant enzymes (CAT, APX, SOD, and POD) in the overexpression lines (Figures 5c,d and 6c,d).These results indicated that the overexpression of the AetPGL gene in A. thaliana increased antioxidant capacity and enhanced tolerance to Cd.

Overexpression of AetPGL Enhanced the Antioxidant Capacity of Transgenic Arabidopsis
We measured the physiological indices of the roots and shoots in both the WT and overexpression lines.The contents of Pro and MDA can serve as indicators to validate plant damage under stressful conditions.In this study, following Cd stress, the MDA content in the WT significantly exceeded that of the transgenic lines (Figures 5a and 6a).Conversely, the Pro content was noticeably lower in the WT compared to the transgenic lines (Figures 5b and 6b), suggesting more severe damage to the cells of WT plants.Furthermore, Cd stress increased the activities of antioxidant enzymes (CAT, APX, SOD, and POD) in the overexpression lines (Figures 5c,d and 6c,d).These results indicated that the overexpression of the AetPGL gene in A. thaliana increased antioxidant capacity and enhanced tolerance to Cd.

Overexpression of AetPGL Enhanced the Antioxidant Capacity of Transgenic Arabidopsis
We measured the physiological indices of the roots and shoots in both the WT and overexpression lines.The contents of Pro and MDA can serve as indicators to validate plant damage under stressful conditions.In this study, following Cd stress, the MDA content in the WT significantly exceeded that of the transgenic lines (Figures 5a and 6a).Conversely, the Pro content was noticeably lower in the WT compared to the transgenic lines (Figures 5b and 6b), suggesting more severe damage to the cells of WT plants.Furthermore, Cd stress increased the activities of antioxidant enzymes (CAT, APX, SOD, and POD) in the overexpression lines (Figure 5c,d and Figure 6c,d).These results indicated that the overexpression of the AetPGL gene in A. thaliana increased antioxidant capacity and enhanced tolerance to Cd.

AetPGL Increased Cadmium Uptake by Regulating the Expression of Heavy Metal Transporters in Roots
We measured the Cd content in OE2 and WT plants after treatment.While there was no difference in the roots, the Cd content in the shoots of the overexpression lines was significantly higher than in the wild type (Figure 7a).The results indicate that overexpression of the AetPGL gene enhances Cd accumulation in the aerial parts.The increased

AetPGL Increased Cadmium Uptake by Regulating the Expression of Heavy Metal Transporters in Roots
We measured the Cd content in OE2 and WT plants after treatment.While there was no difference in the roots, the Cd content in the shoots of the overexpression lines was significantly higher than in the wild type (Figure 7a).The results indicate that overexpression of the AetPGL gene enhances Cd accumulation in the aerial parts.The increased

AetPGL Increased Cadmium Uptake by Regulating the Expression of Heavy Metal Transporters in Roots
We measured the Cd content in OE2 and WT plants after treatment.While there was no difference in the roots, the Cd content in the shoots of the overexpression lines was significantly higher than in the wild type (Figure 7a).The results indicate that overexpression of the AetPGL gene enhances Cd accumulation in the aerial parts.The increased expression levels of the ZIP, IRT, YSL, HMA, and NRAMP genes led to an increase in Cd content in the plants, as their high expression enhances Cd uptake and transport capacity [37].Therefore, we measured the transcript levels of HM transporter-related genes in OE and WT plants.Before and after Cd treatment, the expression level of AtYSL1 in the roots showed no change in either WT or OE plants, but its expression in the shoots of OE plants significantly increased after treatment (Figure 7d,h).Compared to WT, after treatment with 2.5 mM Cd, the transcript levels of AtHMA3, AtZIP1, and AtNRAMP5 were significantly upregulated in the roots of OE2 (Figure 7e-g) and the transcript level of AtHMA3 in the shoots was significantly increased, while the other two genes did not show significant changes (Figure 7i).These results suggest that AetPGL increases Cd content in the aerial parts by upregulating the genes for root transporters in the overexpression lines.
Plants 2024, 13, x FOR PEER REVIEW 9 of 19 expression levels of the ZIP, IRT, YSL, HMA, and NRAMP genes led to an increase in Cd content in the plants, as their high expression enhances Cd uptake and transport capacity [37].Therefore, we measured the transcript levels of HM transporter-related genes in OE and WT plants.Before and after Cd treatment, the expression level of AtYSL1 in the roots showed no change in either WT or OE plants, but its expression in the shoots of OE plants significantly increased after treatment (Figure 7d, h).Compared to WT, after treatment with 2.5 mM Cd, the transcript levels of AtHMA3, AtZIP1, and AtNRAMP5 were significantly upregulated in the roots of OE2 (Figure 7e-g) and the transcript level of AtHMA3 in the shoots was significantly increased, while the other two genes did not show significant changes (Figure 7i).These results suggest that AetPGL increases Cd content in the aerial parts by upregulating the genes for root transporters in the overexpression lines.

AetPGL Promotes Phytohormone Synthesis in Aboveground Parts
To further understand the effect of the AetPGL gene on A. thaliana, we performed transcriptome sequencing and analyzed its regulatory network.A total of 691 DEGs were identified in the Cd-treated WT and transgenic lines (CdOE2 vs. CdWT) (Figure 8a).The results showed that the AetPGL gene affected the transcription of transgenic Arabidopsis after Cd stress.We conducted Gene Ontology (GO) enrichment analysis on the transcriptome data to categorize the biological functions of DEGs identified in CdOE2 and CdWT.We found that these DEGs were mainly enriched in the cellular anatomical entity, cellular processes, binding, and responses to stimulus (Figure 8b).Most of these biological

AetPGL Promotes Phytohormone Synthesis in Aboveground Parts
To further understand the effect of the AetPGL gene on A. thaliana, we performed transcriptome sequencing and analyzed its regulatory network.A total of 691 DEGs were identified in the Cd-treated WT and transgenic lines (CdOE2 vs. CdWT) (Figure 8a).The results showed that the AetPGL gene affected the transcription of transgenic Arabidopsis after Cd stress.We conducted Gene Ontology (GO) enrichment analysis on the transcriptome data to categorize the biological functions of DEGs identified in CdOE2 and CdWT.We found that these DEGs were mainly enriched in the cellular anatomical entity, cellular processes, binding, and responses to stimulus (Figure 8b).Most of these biological processes are related to cell molecular activity and response to stress.In the KEGG pathway, DEGs are mainly enriched in metabolic pathways, biosynthesis of secondary metabolites, and glutathione and plant hormone signal transduction pathways (Figure 8c).
Prior to Cd treatment, there were no differences in hormone content between the WT and OE lines.However, after treatment with 2.5 mM Cd, the BR, GA, and JA in both WT and OE2 lines exhibited an increasing trend, and significant differences were observed, although CK showed no difference (Figure 10a-d).Under Cd stress, all hormone levels significantly increased in both WT and transgenic plants.Importantly, the levels of GA, JA, and BR in transgenic plants were significantly higher than in the WT.These results indicate that AetPGL may enhance the Cd tolerance of transgenic Arabidopsis by regulating these plant hormones.Notably, we observed significant changes in the expression levels of key genes involved in plant hormone signal transduction (Table A2).Subsequently, we analyzed this pathway and identified two, one, two, and four genes that were significantly upregulated in the synthesis pathways of brassinosteroids, gibberellin acid, jasmonic acid, and cytokinin, respectively (Figure 8d).We selected eight hormone-related genes for RT-qPCR verification, and also verified the consistency of the transcriptome data (Figure 9).Following this, we determined the contents of CK, GA, BR, and JA in both WT and transgenic plants.Prior to Cd treatment, there were no differences in hormone content between the WT and OE lines.However, after treatment with 2.5 mM Cd, the BR, GA, and JA in both WT and OE2 lines exhibited an increasing trend, and significant differences were observed, although CK showed no difference (Figure 10a-d).Under Cd stress, all hormone levels significantly increased in both WT and transgenic plants.Importantly, the levels of GA, JA, and BR in transgenic plants were significantly higher than in the WT.These results indicate that AetPGL may enhance the Cd tolerance of transgenic Arabidopsis by regulating these plant hormones.

Discussion
PGL, as one of the key enzymes in the OPPP pathway, serves as a crucial source of NADPH in plant organisms, maintaining the balance of NADPH within the plant.Current research indicates that NADPH is considered the most important molecule determining the potential antioxidant capacity of cells.When plants undergo oxidative stress, more NADPH is required to maintain normal redox status [38].ROS is a metabolite that plays a crucial role in regulating plant growth and development.It is considered to be a significant mediator involved in plant signal transduction, contributing greatly to the growth and development of plants at various stages and their responses to various environmental stresses [39].The levels of ROS are determined by a tightly controlled balance between production and decomposition, achieved through complex and highly intricate antioxidant systems.However, excessive ROS can have toxic effects on plants, leading to rapid cell death [40].As the final product of membrane lipid peroxidation, the content of MDA will increase with the damage of membrane lipids, which also reflects the more serious cell damage.Pro can regulate the permeability of membrane lipids, prevent cells from being overoxidized, and reflect the antioxidant capacity of plants [41,42].Plants have developed a unique antioxidant system under stress conditions, and SOD, POD, APX, and CAT are considered crucial scavengers of ROS and integral components of plant antioxidant defenses [43].In this study, WT plants experienced more severe peroxidation damage, while transgenic plants overexpressing AetPGL exhibited a stronger antioxidant capacity under Cd stress (Figures 5c-f and 6c-f).In comparison to the WT, Cd stress was less toxic to transgenic plants, resulting in a significant reduction in MDA content (Figures 5a and 6a).However, Pro levels increased significantly (Figures 5b and 6b), indicating an enhanced response to oxidative stress in the transgenic plants.
The absorption of Cd from the soil and its transport within plants depend on the involvement of various transport proteins.Numerous studies have reported that multiple transporters, including IRT, ZIP, NRAMP, and HMA, participate in the uptake and transport of heavy metals, making them critical in heavy metal detoxification.In this study, it was found that the Cd content in the leaves of OE2 transgenic Arabidopsis was significantly higher than in the WT, with no significant difference in the roots, as shown in Figure 7a.Under Cd treatment, the Cd content in the stems of transgenic plants was more than 75.86% higher than in the WT, suggesting that AetPGL may enhance heavy

Discussion
PGL, as one of the key enzymes in the OPPP pathway, serves as a crucial source of NADPH in plant organisms, maintaining the balance of NADPH within the plant.Current research indicates that NADPH is considered the most important molecule determining the potential antioxidant capacity of cells.When plants undergo oxidative stress, more NADPH is required to maintain normal redox status [38].ROS is a metabolite that plays a crucial role in regulating plant growth and development.It is considered to be a significant mediator involved in plant signal transduction, contributing greatly to the growth and development of plants at various stages and their responses to various environmental stresses [39].The levels of ROS are determined by a tightly controlled balance between production and decomposition, achieved through complex and highly intricate antioxidant systems.However, excessive ROS can have toxic effects on plants, leading to rapid cell death [40].As the final product of membrane lipid peroxidation, the content of MDA will increase with the damage of membrane lipids, which also reflects the more serious cell damage.Pro can regulate the permeability of membrane lipids, prevent cells from being overoxidized, and reflect the antioxidant capacity of plants [41,42].Plants have developed a unique antioxidant system under stress conditions, and SOD, POD, APX, and CAT are considered crucial scavengers of ROS and integral components of plant antioxidant defenses [43].In this study, WT plants experienced more severe peroxidation damage, while transgenic plants overexpressing AetPGL exhibited a stronger antioxidant capacity under Cd stress (Figures 5c-f and 6c-f).In comparison to the WT, Cd stress was less toxic to transgenic plants, resulting in a significant reduction in MDA content (Figures 5a and 6a).However, Pro levels increased significantly (Figures 5b and 6b), indicating an enhanced response to oxidative stress in the transgenic plants.
The absorption of Cd from the soil and its transport within plants depend on the involvement of various transport proteins.Numerous studies have reported that multiple transporters, including IRT, ZIP, NRAMP, and HMA, participate in the uptake and transport of heavy metals, making them critical in heavy metal detoxification.In this study, it was found that the Cd content in the leaves of OE2 transgenic Arabidopsis was significantly higher than in the WT, with no significant difference in the roots, as shown in Figure 7a.Under Cd treatment, the Cd content in the stems of transgenic plants was more than 75.86% higher than in the WT, suggesting that AetPGL may enhance heavy metal resistance in Arabidopsis by regulating transporters to sequester Cd in vacuoles within the leaves.HMA, as a major transporter of heavy metals, plays a crucial role in plant heavy metal resistance.We observed that the expression level of AtHMA3 in both the aerial and root parts of OE lines was significantly higher than in the WT after Cd treatment.In Arabidopsis, AtHMA3 is primarily localized on the vacuolar membrane, where its main function is to transport Cd into vacuoles for sequestration, thereby reducing the toxic effects on other organelles.Overexpression of AtHMA3 has been shown to lead to the accumulation of more Cd in roots and stems [44].In rice, OsHMA3 is a key determinant of heavy metal accumulation in grains, as it is highly expressed in roots and sequesters Cd into vacuoles [45].In this study, we found that after treatment, the expression level of AtHMA3 in both the shoots and roots of OE lines was significantly elevated.This may be one of the reasons why the Cd content in the shoots was higher than in the WT.However, in the roots, although the expression level of AtHMA3 increased, the Cd content did not show a significant change compared to the WT.We speculate that this could be due to the combined effects of AtHMA3 with other metal transport proteins [46].NRAMP5 primarily functions in the roots, and in this study, we observed that its expression level in the roots of OE lines was significantly increased after treatment.However, the specific function of AtNRAMP5 has not yet been fully studied.In rice, OsNRAMP5, a homolog of AtNRAMP5, is located on the plasma membrane and, when highly expressed in the roots, transports Cd and Mn into the stems, leading to increased Cd accumulation in the stems.Knockout lines of OsNRAMP5 showed lower levels of Mn and Cd in roots and stems compared to control lines, indicating a loss of the ability to uptake Mn and Cd [47].IRT is another key transporter, and ectopic expression of AtIRT1 in Solanum nigrum enhanced antioxidant capacity and increased Cd accumulation by 19% compared to control groups [48].The increased expression of IRT1 induced by soil iron availability promotes Cd accumulation and transport in dicotyledonous vegetables [49].However, in this study, the OE lines did not show a significant increase in expression levels compared to the WT, suggesting that AetPGL may not regulate IRT proteins.In contrast, the suppression of BcIRT1 and BcZIP2 expression levels reduced Cd uptake in Brassica chinensis roots [50].AtZIP1, as an important transporter, primarily transports divalent metal ions in plants and can move Cd from the root to the shoot.Studies have shown that increased ZIP1 expression promotes Cd accumulation and transport in both Arabidopsis and maize [51,52].In this study, after Cd treatment, the expression level of AtZIP1 was upregulated in the roots of OE lines, but there was no significant change in the shoots, while the expression level in the WT showed no significant change before and after treatment.This indicates that the expression of AetPGL may lead to changes in ZIP1, enhancing the transport of Cd to the shoots.In this study, the expression of genes encoding heavy metal transporters in the roots of the transgenic lines was significantly higher than in the WT lines (Figure 7c-e), which enhanced the transport of Cd from roots to stems.
As endogenous substances, plant hormones play a crucial role in regulating plant growth and development, particularly at extremely low concentrations, with complex and intricate functions.Plant hormones, acting as signal transduction biomolecules, typically operate in small quantities, extensively regulating the physiological and biochemical mechanisms of plants.Moreover, they play a key role in resisting external stress [53,54].Additionally, genes such as AtAHP4, AtARR11, AtRGL1, AtBES1, AtTCH4, AtTIFY7, At-TIFY10B, and AtJAZ3, all participating in hormone synthesis (including cytokinin, jasmonic acid, gibberellin, and brassinosteroids), were upregulated under Cd stress (Figure 9a-h).
Prior studies have indicated that the application of plant hormones on plant leaves can effectively alleviate toxicity and enhance plant tolerance to HMs.Furthermore, when plants are stressed by HMs, their endogenous plant hormones undergo immediate changes to cope with the toxic effects and mitigate damage [55,56].Brassinosteroids are hormones capable of regulating the absorption of ions by plant cells, thereby significantly reducing the accumulation of heavy metals.Cd, even at lower concentrations, can induce harmful effects in plants by hindering chlorophyll biosynthesis and the expression of enzymes [57].Researchers have discovered that another brassinosteroid, BR-epibrassinolide (EPL), reduces Cd-induced oxidative burst by upregulating the expression of antioxidant enzymes and various stresses signaling hormones, particularly in photosynthetic pigments and cotyledons [58].GA not only promotes the normal growth and development of plants but also participates in the detoxification of heavy metals [59].Protein and nitrogen content are key factors for stress resistance and adaptation.In a previous study on peas, 10 µM GA improved seed germination and stem elongation in chromium-stressed pea plants by increasing protein and nitrogen content [60].Additionally, GA treatment of pepper plants can modulate their endogenous iron homeostasis mechanism, slowing down overall plant growth to protect, prevent, or adapt to cadmium stress [61].In fava beans, JA alleviates the detrimental effects of Cd stress by enhancing antioxidant enzyme activity and reducing the deposition of Cd, hydrogen peroxide (H 2 O 2 ), and MDA in plant tissues [62].Additionally, the addition of JA to rapeseed plants reduces MDA concentration and Cd uptake in leaves, leading to increased tolerance to HM stress in rapeseed seedlings under Cd stress [63].Moreover, it has been reported that the exogenous application of MeJA (1, 5, and 10 µM) can enhance the growth of peas under Cd stress [64].Cytokinin is not only involved in cell division and plant morphogenesis but also plays a role in the stress response to HMs.After Cd stress, cytokinin signal transduction in A. thaliana is activated, and overexpression in roots results in the significant upregulation of genes related to cytokinin synthesis, including AtARR1, AtARR12, and AtAHK3, to cope with the damage caused by Cd stress [65].Furthermore, the foliar application of CK and ABA substantially mitigates the toxic effects of cobalt (Co) in tomato seedlings by modulating the absorption, transport, and chelation of Co in plant tissues [66].In the study, the contents of BR, GA, and JA were significantly different after treatment, and the content of OE2 was significantly higher than that of the WT (Figure 10a-c).The results showed that the AetPGL gene reduced the harm of Cd stress by regulating plant hormones.

Conclusions
In this study, the AetPGL gene was identified from Ae. tauschii and found to be responsive to Cd stress.The ectopic expression of AetPGL enhanced Cd tolerance by regulating the activities of antioxidant enzymes.Furthermore, it increased Cd accumulation in A. thaliana by influencing the expression of heavy metal transporters such as AtHMA3, AtNRAMP5, and AtIRT1 in the roots.Transcriptome analysis showed that DEGs were involved in plant hormone signal transduction pathways along with the increase in plant hormone levels (including BR, GA, JA, and CK) under Cd stress.These results are helpful to reveal the tolerance mechanism of plants to Cd and provide a theoretical basis for molecular breeding in crops with low Cd accumulation.Funding: This research was funded by the National Natural Science Foundation of China, grant number 32260506 and the earmarked fund for GZMARS-Rapeseed.

Figure 1 .
Figure 1.Evolutionary relationships (a) and sequence analysis (b) between relevant PGL proteins.

Figure 2 .
Figure 2. Expression patterns of the AetPGL gene.(a) Relative expression of AetPGL under Cd stress in Aegilops tauschii.(b) Relative expression of AetPGL in wild-type (WT) and transgenic Arabidopsis overexpressing the AetPGL gene.Different colors represent different overexpression lines.Different letters indicate a statistically significant difference at p < 0.05.

Figure 1 .
Figure 1.Evolutionary relationships (a) and sequence analysis (b) between relevant PGL proteins.

Figure 1 .
Figure 1.Evolutionary relationships (a) and sequence analysis (b) between relevant PGL proteins.

Figure 2 .
Figure 2. Expression patterns of the AetPGL gene.(a) Relative expression of AetPGL under Cd stress in Aegilops tauschii.(b) Relative expression of AetPGL in wild-type (WT) and transgenic Arabidopsis overexpressing the AetPGL gene.Different colors represent different overexpression lines.Different letters indicate a statistically significant difference at p < 0.05.

Figure 2 .
Figure 2. Expression patterns of the AetPGL gene.(a) Relative expression of AetPGL under Cd stress in Aegilops tauschii.(b) Relative expression of AetPGL in wild-type (WT) and transgenic Arabidopsis overexpressing the AetPGL gene.Different colors represent different overexpression lines.Different letters indicate a statistically significant difference at p < 0.05.

Figure 3 .
Figure 3.The growth state of Arabidopsis overexpressing the AetPGL gene and WT plants under Cd stress.(a-b) Root length and germination rate of transgenic Arabidopsis grown for 14 days on halfstrength Murashige and Skoog (1/2 MS) medium with or without 100 µM and 150 µM Cd.Scale bar = 1.5 cm.(c) Stem morphology of wild-type and transgenic Arabidopsis treated with 0 or 2.5 mM Cd for 14 days.Scale bar = 5 cm.

Figure 4 .
Figure 4.The root length and germination rate of WT and transgenic Arabidopsis were cultured on half-strength mouse and Skoog (1/2 MS) medium without or with 100 µM and 150 µM Cd for 14 days.(a) Root lengths of WT and transgenic lines before and after treatment.(b) Germination rates of WT and transgenic lines before and after treatment.Different letters indicate a statistically significant difference at p < 0.05.

Figure 3 .
Figure 3.The growth state of Arabidopsis overexpressing the AetPGL gene and WT plants under Cd stress.(a,b) Root length and germination rate of transgenic Arabidopsis grown for 14 days on half-strength Murashige and Skoog (1/2 MS) medium with or without 100 µM and 150 µM Cd.Scale bar = 1.5 cm.(c) Stem morphology of wild-type and transgenic Arabidopsis treated with 0 or 2.5 mM Cd for 14 days.Scale bar = 5 cm.

Figure 3 .
Figure 3.The growth state of Arabidopsis overexpressing the AetPGL gene and WT plants under Cd stress.(a-b) Root length and germination rate of transgenic Arabidopsis grown for 14 days on halfstrength Murashige and Skoog (1/2 MS) medium with or without 100 µM and 150 µM Cd.Scale bar = 1.5 cm.(c) Stem morphology of wild-type and transgenic Arabidopsis treated with 0 or 2.5 mM Cd for 14 days.Scale bar = 5 cm.

Figure 4 .
Figure 4.The root length and germination rate of WT and transgenic Arabidopsis were cultured on half-strength mouse and Skoog (1/2 MS) medium without or with 100 µM and 150 µM Cd for 14 days.(a) Root lengths of WT and transgenic lines before and after treatment.(b) Germination rates of WT and transgenic lines before and after treatment.Different letters indicate a statistically significant difference at p < 0.05.

Figure 4 .
Figure 4.The root length and germination rate of WT and transgenic Arabidopsis were cultured on half-strength mouse and Skoog (1/2 MS) medium without or with 100 µM and 150 µM Cd for 14 days.(a) Root lengths of WT and transgenic lines before and after treatment.(b) Germination rates of WT and transgenic lines before and after treatment.Different letters indicate a statistically significant difference at p < 0.05.

Figure 7 .
Figure 7. (a) Cd concentration in the roots and shoots of WT and transgenic Arabidopsis plants overexpressing AetPGL (OE2) after a 14-day treatment with 2.5 mM Cd.(b) Expression level of AtNRAMP5 in the root, (c) expression level of AtIRT1 in the shoot, (d-g) expression levels in the root for AtHMA3, AtYSL1, AtZIP1, and AtNRAMP5.(h-k) The expression levels in the shoot for AtHMA3, AtYSL1, AtZIP1, and AtNRAMP5.Different letters indicate statistically significant differences at p < 0.05.

Figure 7 .
Figure 7. (a) Cd concentration in the roots and shoots of WT and transgenic Arabidopsis plants overexpressing AetPGL (OE2) after a 14-day treatment with 2.5 mM Cd.(b) Expression level of AtNRAMP5 in the root, (c) expression level of AtIRT1 in the shoot, (d-g) expression levels in the root for AtHMA3, AtYSL1, AtZIP1, and AtNRAMP5.(h-k) The expression levels in the shoot for AtHMA3, AtYSL1, AtZIP1, and AtNRAMP5.Different letters indicate statistically significant differences at p < 0.05.

Figure 8 .
Figure 8. Transcriptome analysis of DEGs in the shoots of WT and OE2 plants overexpressing AetPGL under Cd stress.(a) Number of DEGs.(b) Gene Ontology (GO) classification of DEGs.(c) KEGG pathway enrichment of DEGs.(d) Expression of DEGs in the plant hormone signal transduction pathway.