Next Article in Journal
Mechanistic Insights into the Anti-Proliferative Action of Gut Microbial Metabolites against Breast Adenocarcinoma Cells
Next Article in Special Issue
Transcriptome Profiling, Physiological and Biochemical Analyses Reveal Comprehensive Insights in Cadmium Stress in Brassica carinata L.
Previous Article in Journal
Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from β-Thalassemia Patients Treated with Sirolimus
Previous Article in Special Issue
Transcriptome Sequencing Analysis of Root in Soybean Responding to Mn Poisoning
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 20
10.3390/ijms242015052
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Overexpression of ApHIPP26 from the Hyperaccumulator Arabis paniculata Confers Enhanced Cadmium Tolerance and Accumulation to Arabidopsis thaliana

by
Lizhou Zhou
†,
Lvlan Ye
†,
Biao Pang
,
Yunyan Hou
,
Junxing Yu
,
Xuye Du
,
Lei Gu
,
Hongcheng Wang
* and
Bin Zhu
*
School of Life Sciences, Guizhou Normal University, Guiyang 550025, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(20), 15052; https://doi.org/10.3390/ijms242015052
Submission received: 11 September 2023 / Revised: 5 October 2023 / Accepted: 9 October 2023 / Published: 10 October 2023
(This article belongs to the Special Issue Plant Responses to Heavy Metals: From Deficiency to Excess)
Browse Figures
Review Reports Versions Notes

Abstract

:
Cadmium (Cd) is a toxic heavy metal that seriously affects metabolism after accumulation in plants, and it also causes adverse effects on humans through the food chain. The HIPP gene family has been shown to be highly tolerant to Cd stress due to its special domain and molecular structure. This study described the Cd-induced gene ApHIPP26 from the hyperaccumulator Arabis paniculata. Its subcellular localization showed that ApHIPP26 was located in the nucleus. Transgenic Arabidopsis overexpressing ApHIPP26 exhibited a significant increase in main root length and fresh weight under Cd stress. Compared with wild-type lines, Cd accumulated much more in transgenic Arabidopsis both aboveground and underground. Under Cd stress, the expression of genes related to the absorption and transport of heavy metals underwent different changes in parallel, which were involved in the accumulation and distribution of Cd in plants, such as AtNRAMP6 and AtNRAMP3. Under Cd stress, the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) in the transgenic lines were higher than those in the wild type. The physiological and biochemical indices showed that the proline and chlorophyll contents in the transgenic lines increased significantly after Cd treatment, while the malondialdehyde (MDA) content decreased. In addition, the gene expression profile analysis showed that ApHIPP26 improved the tolerance of Arabidopsis to Cd by regulating the changes of related genes in plant hormone signal transduction pathway. In conclusion, ApHIPP26 plays an important role in cadmium tolerance by alleviating oxidative stress and regulating plant hormones, which provides a basis for understanding the molecular mechanism of cadmium tolerance in plants and provides new insights for phytoremediation in Cd-contaminated areas.

1. Introduction

Drought, extreme temperature, high salinity and heavy metals are the most serious abiotic stresses affecting plant growth [1]. Heavy metals (HMs) are one of the most critical abiotic factors affecting soil and water and have become a serious worldwide problem [2]. They are considered to be the most serious pollutant in ecotoxicology. Although HMs exist in nature, due to the rapid development of industry their content is increasing, resulting in great adverse effects [3,4]. Cadmium (Cd) is a nonessential element for plants and has become one of the most toxic pollutants in water and soil worldwide [5]. It not only causes soil dysfunction and water quality decline but also damages the physiological development of plants to varying degrees, such as reducing chlorophyll, inhibiting nutrient absorption, and causing cell death, thus affecting the growth and metabolism of plants [6,7,8,9]. Cd is absorbed by plants, enriched and transferred into the food chain, endangering human life and health [10,11,12]. Considering the threat posed by Cd to crop production and quality as well as human and animal health, reducing its accumulation is essential. Therefore, it has been an urgent and high-priority issue to elucidate the molecular mechanism of Cd tolerance in plants and to breed or engineer crops with higher tolerance but less accumulation of Cd.
Because Cd has a similar structure to other essential elements (Ca, Zn, and Fe), the transporter transports these essential elements while transporting Cd into the plant. As a heavy metal, a large amount of Cd accumulation will have a toxic effect on the plant. [12,13]. Heavy metals enter the roots of plants from the soil through complex pathways and are then transported to the aboveground parts. This process involves the participation of some heavy metal-related transporters, including iron-regulated transporter (IRT), zinc-regulated transporter/IRT-like protein (ZIP), natural resistance-associated macrophage protein (NRAMP) and heavy metal ATPase (HMA) [14,15].
HIPPs (heavy metal-associated isoprenylated plant proteins) are unique metallochaperone proteins in vascular plants and are mainly involved in the reaction of heavy metal homeostasis, detoxification mechanisms, heavy metal tolerance (especially Cd tolerance), cold resistance, drought resistance and other mechanisms [16]. These proteins generally contain 113 to 584 amino acid residues and have one or two heavy metal-associated domains (HMAs), of which the core motif is M/LXCXXC (M for methionine, L for leucine, X for amino acids, C for cysteine), with the ability to bind to Cu, Ni, and Zn [17]. A typical structural feature of HIPPs is that the C-terminus has a CaaX structure (C represents cysteine; a represents aliphatic amino acids; X represents methionine, glutamine, cysteine, serine, or alanine), which is crucial for biological functions with protein–membrane and protein-protein interactions [18]. The function of HIPPs is apparent but only characterized in a handful of plants. Whole-genome analysis showed 56, 54 and 13 HIPP genes in Camellia sinensis (L.) Kuntze [19], Oryza sativa L. [20], and Fagopyrum tataricum (L.) Gaertn. [21], respectively. CsHIPP24 overexpressed in Cd-sensitive yeast (ycf1) displayed higher performance than CsHIPP22/36 in Cd resistance. Overexpression of the CdI19 gene enhances the blocking effect of the endoplasmic reticulum and reduces the damage of cadmium to cells in Arabidopsis [22]. The overexpression of OsHIPP56 exhibited a remarkable detoxification effect with an up to 230% reduction in Cd deposition in rice roots compared with WT [23]. The expression level of OsHIPP21 was increased by Cd induction, as was the case for OsHIPP28 and OsHIPP41 [24]. The transformed yeast mutants expressing OsHIPP16/34/60 accumulated more heavy metals (treated with Mn, Cu, Cd and Zn, respectively) without growth inhibition, which was validated by rice transformation of OsHIPP42 [18]. AtHIPP44 can interact with the transcription factor AtMYB49 and increase its expression, thereby reducing Cd accumulation [25]. Although there are many reviews on HIPPs, the functional verification of HIPP26 is lacking.
It has been reported that Arabis paniculata Franch is a new type of heavy metal hyperaccumulator that has a strong enrichment effect on Zn, Cd and Pb [26]. In our previous study, we sequenced the transcriptome of A. paniculata under Cd stress and found that the HIPP26 gene was significantly upregulated [27]. However, the role of this gene in the physiological response and molecular mechanism to Cd stress is unclear. Therefore, the ApHIPP26 gene was cloned and overexpressed in Arabidopsis thaliana (L.) Heynh to verify the molecular mechanism under Cd stress. Overexpression of ApHIPP26 enhanced Cd tolerance and accumulation by regulating plant hormone signal transduction in A. thaliana.

2. Results

2.1. ApHIPP26 Conserved Motif and Phylogenetic Analysis

The ApHIPP26 protein was conserved among the 10 species of plant. As shown in Figure 1a, the ApHIPP26 protein is evolutionarily close to that of C. sativa. Motif analysis showed that the motif of ApHIPP26 is overwhelmingly identical to that of ApHIPP26 proteins in other plant species (Figure 1b).

2.2. ApHIPP26 Is Localized in the Nucleus

To determine the subcellular localization of ApHIPP26, we injected ApHIPP26-GFP and GFP expression vectors into tobacco epidermal cells for transient expression. The GFP signal and DAPI signal completely overlapped, while the negative control signal was distributed in the cell membrane, cytoplasm and nucleus (Figure 2). These results indicate that ApHIPP26 is localized in the nucleus.

2.3. Cd Induced ApHIPP26 Expression

RT-qPCR results showed that there was no difference in the expression level of ApHIPP26 in roots and shoots of A. paniculata under normal conditions. After exposure to Cd, the mRNA levels of roots and shoots increased significantly, especially in the latter part (Figure 3a). The results show that the ApHIPP26 gene is a key gene of A. paniculata in response to Cd stress, and its expression is upregulated when Cd stress occurs.

2.4. ApHIPP26 Overexpression Enhanced Cd Tolerance and Accumulation in Transgenic Arabidopsis

We obtained nine transgenic Arabidopsis lines by inflorescence infection, and fluorescence quantitative RT-qPCR was performed to detect ApHIPP26 expression in these nine lines. Three lines (OE2, OE5 and OE9) with high expression levels were options for further operation (Figure 3b). In a normal medium, there was no significant difference in the phenotype (root length, fresh weight, germination rate) of wild-type (WT) and transgenic lines. However, in the medium with 100 μM Cd, the root length of the wild type (WT) was significantly shorter than that of the three transgenic lines with the same performance in both fresh weight and germination rate, of which the wild type was significantly lower (Figure 4a,b and Figure 5a–c). The results of 14-day soil incubation showed no difference in the phenotypes of WT and transgenic lines under normal conditions, but the leaves of WT showed much more severe yellowness than those of transgenic plants treated with 2.5 mM Cd (Figure 4c), which indicated that the overexpression of ApHIPP26 enhanced Cd tolerance in transgenic Arabidopsis.

2.5. Overexpression of ApHIPP26 Enhanced the Antioxidant Capacity and Photosynthesis of Transgenic Arabidopsis

We detected the contents of MDA and proline (Pro), which can reflect the degree of plant injury under stress. In this study, the MDA content of WT plants was significantly higher than that of transgenic lines stressed by Cd (Figure 6a), and the content of proline in WT plants showed a downwards trend compared to transgenic plants (Figure 6b), indicating that the cell membrane of WT plants had suffered more serious damage. Meanwhile, the antioxidant system in the plant functioned, eliminating some ROS to mitigate the effects of stress. The POD, APX, CAT, and SOD enzymes in OE plants exhibited significantly higher activity than those in WT plants (Figure 6c–f). Moreover, the chlorophyll content was rescued in transgenic plants and was significantly higher than that in WT plants under Cd stress (Figure 6g). These results implied that overexpression of ApHIPP26 in Arabidopsis enhanced Cd tolerance by improving the antioxidant capacity and rescuing photosynthesis.

2.6. ApHIPP26 Increased Cd Uptake by Regulating the Expression of Heavy Metal Transporters

After Cd exposure, the Cd content of shoots and roots in the OE5 and WT lines indicated that overexpression of ApHIPP26 increased Cd uptake both aboveground and underground (Figure 7i).
Plants have developed a regulatory system to maintain the balance of metal ion concentrations, among which heavy metal transporters are an important component and include the yellow stripe-like transporter protein family (YSL), ZRT/IRT-like protein family (ZIP), natural resistance-associated macrophage protein (NRAMP), etc. We selected eight genes encoding related proteins to verify whether overexpression of ApHIPP26 regulates their expression. In the absence of cadmium treatment, overexpressed ApHIPP26 elevated the expression level of AtNRAMP6 and AtNAS3 and decreased that of AtHMA2, with no difference in others. Under Cd treatment, AtNRAMP6 and AtNAS3 were expressed at significantly higher levels, and AtNRAMP3, AtYLS2, AtIRT3, AtZIP4, and AtCAX3 were downregulated.

2.7. ApHIPP26 Enhanced the Synthesis of Plant Hormones in the Aerial Parts of Transgenic A. thaliana

To further understand the regulatory network after the ApHIPP26 gene was introduced into A. thaliana, WT and OE5 were treated with 0 mM and 2.5 mM, and the DEGs of their aboveground parts were identified. There were 223 DEGs in transgenic lines in the comparison with Cd treatment and without (CKOE5/CdOE5), and 4652 EDGs in WT lines treated with and without Cd (CKWT/CdWT). In addition, a total of 2928 DEGs were identified in Cd-treated WT and transgenic lines (CdOE5/CdWT), while 1217 DEGs were identified in the control group without Cd treatment (CKOE5/CKWT). The results showed that the ApHIPP26 gene altered the transcription profile of the Cd stress response after its transfer into Arabidopsis (Figure 8a).
Subsequently, we performed GO analysis to classify the biological functions of the DEGs identified in CdOE5 vs CdWT. We found that many of these DEGs were enriched in the lignin biosynthetic process, cell wall macromolecule biosynthetic process, and plant-type cell wall modification (Figure 8b). These biological processes are closely related to plant cell wall synthesis, and the DEGs were all downregulated. In the KEGG pathway, DEGs were mainly enriched in the starch and sucrose metabolism, plant hormone signal transduction, and cysteine and methionine metabolism pathways (Figure 8c). Noticeably, we found that the expression levels of DEGs in the plant hormone signal transduction pathway changed significantly (Supplementary Tables S2–S4), so we analysed them (Figure 8d). Twenty, ten and three DEGs were detected in auxin, abscisic acid (ABA) and ethylene (ETH) signal transduction, respectively. AtIAA27 and AtARF11 were upregulated, and AtHAB1, AtABI2 and AtERF1 were downregulated (Figure 9a–h).

3. Discussion

Cd can pollute soil, water, air dust and food in various ways, which causes Cd poisoning in people and animals, mainly by feeding or inhalation, and cadmium compounds are the most severe. Cd poisoning mainly manifests as kidney damage, osteoporosis, anemia, nervous system damage, etc., and severe cases can lead to kidney failure and death. Human chromosome aberrations caused by chronic poisoning seriously affect offspring growth and development [28]. To date, many technologies for the remediation of heavy metal-contaminated soil are mainly based on physical and chemical principles [29,30,31]. However, some limitations remain, such as high cost and low efficiency when pollutants are present at low concentrations [31,32,33]. Therefore, it is necessary to develop more economical and efficient technologies. Phytoremediation is plant-based remediation that can absorb metallic elements from soil and water, accumulate them in plants and purify the environment [34]. Recently, the study of hyperaccumulators has shed new light on these issues of heavy metal pollution and has become one of the research highlights.
According to professional evaluation and analysis, many star plants applied in phytoremediation have been found, and molecular mechanisms and useful candidate genes for the absorption and accumulation of heavy metals have been explored in these plants. In the Populus×canescens line, the CTP3 gene from Sedum plumbizincicola X. H. Guo was overexpressed and increased cadmium tolerance and accumulation, altering the distribution of Cd in leaf tissues [35]. Moreover, RNAi and overexpression techniques proved that SpHMA3 positively regulated Cd accumulation by compartmentalization in Sedum plumbizincicola [36]. Overexpression of the root-specific expression gene PCR2 in Sedum alfredii Hance enhanced the Cd exocytosis of roots, which was confirmed in A. thaliana [37]. Ectopic expression of ZNT1 cloned from Noccaea caerulescens (J.Presl & C.Presl) F.K.Mey. revealed that NcZNT1 contributed greatly to root-shoot transport of metals such as nickel, Cd, zinc, iron (Fe), and manganese (Mn) in A. thaliana [38]. The upwards transport of Fe and Mn rather than Cu or Zn was increased in transgenic A. thaliana overexpressing SnYSL3 (Solanum nigrum L.), but when stressed by Cd the transport efficiency of Mn no longer changed [39]. MsYSL1, one upregulated gene in Miscanthus sacchariflorus treated with Cd, manages the transport of Cd, Fe, and Mn and is involved in the process of Cd detoxification by inducing the expression of genes related to nicotianamine synthesis and HM exclusion [40]. Previously, we executed one transcriptome study and found that the ApHIPP26 gene in the hyperaccumulator A. paniculata is induced by Cd stress [41]. In this study, we ectopically expressed ApHIPP26 in Arabidopsis to further explore its function in the response to HM stress.
Reactive oxygen species (ROS), secondary metabolites produced in aerobic metabolism, are recognized as key signal transduction molecules that are deeply involved in programmed cell death, growth and development, hormone signaling, and responses to various environmental stresses [42]. However, excessive ROS mainly attack biological macromolecules (membrane lipids, proteins and nucleic acids) within cells, producing cytotoxic effects and even triggering rapid cell death [43]. The generation and digestion of ROS in cells must remain in dynamic equilibrium, which is the basis of maintaining a relatively stable redox state for survival. As the final product of membrane lipid peroxidation, MDA content increases with increasing lipid membrane oxidation damage, and Pro can regulate permeability and prevent peroxidation, reflecting the antioxidant capacity of plants [44]. SOD, POD, and CAT are considered important scavengers and components of antioxidant defense in plants [45]. In this study, transgenic plants overexpressing ApHIPP26 exhibited stronger antioxidant capacity under Cd stress, with excellent antioxidant oxidase activity (Figure 6), and the content of MDA significantly decreased, while the WT plants suffered more seriously from peroxide. Of course, overexpression of ApHIPP26 also increased root growth and biomass, which is important for plant growth (Figure 5).
The most important feature of hyperaccumulator plants is their ability to absorb heavy metals and transfer them to aerial parts [46]. In this study, overexpression of ApHIPP26 significantly increased the uptake and translocation of Cd in Arabidopsis (Figure 7b). Therefore, we detected some genes known to be involved in the above processes and found that the expression of AtNRAMP6 and AtNAS3 was significantly upregulated, while that of AtNRAMP3, AtYLS2, AtIRT3, AtZIP4, and AtCAX3 was downregulated. SaNRAMP6 from Sedum alfredii, when transferred to yeast and Arabidopsis, enhances their sensitivity to Cd by increasing Cd transportation and accumulation [47]. In tobacco, knocking out the NtNRAMP3 gene makes it easier for Cd to accumulate in vacuoles, altering the distribution of Cd in subcellular cells of leaves [48]. Due to the overexpression of ApHIPP26, the expression of genes related to heavy metal absorption and transport undergoes different changes in parallel, jointly participating in the accumulation and distribution of Cd in plants. This also confirms that overexpression of ApHIPP26 enhances cadmium accumulation.
In hyperaccumulators, the main transport site of metals is the xylem, in which heavy metals form complexes with various organic acids or amino acids and are translocated upwards [49]. In this situation, the metal ions deposited in the cell wall are not easily transported [50]. After entering leaf tissues, the metal mainly crossed the cell wall and became compartmentalized in the vacuole [2]. Our GO analysis revealed that DEGs are mainly classified into cell wall synthesis and are all downregulated, which makes it easier for heavy metals to enter rather than bind to the cell wall. Additionally, our KEGG analysis revealed that DEGs were enriched in cysteine and methionine metabolism pathways (Figure 8c). As reported, sulfur-containing methionine and cysteine achieve transport and detoxification of heavy metals through phytochelatins and metallothioneins, and these chelates and complexes can reduce the toxicity of heavy metals and ROS by the oxidation reaction of their thiol group (-SH) [51,52]. Exogenous cysteine can improve the tolerance of plants (maize, Arabidopsis) to heavy metals such as Cd, chromium and mercury by increasing the activity of antioxidant enzymes and regulating the expression of GS1 and MT3 genes, which encode glutathione and metallothioneins [53]. Precursors involved in cysteine and methionine biosynthesis are also closely related to abiotic stress in plants. The CSase gene, belonging to the cysteine synthase gene family and involved in cysteine synthesis, was overexpressed in Medicago sativa and significantly increased the content of cysteine and glutathione, which contributed to Cd resistance in alfalfa [54]. SAMS1, one gene encoding S-adenosyl-methionine synthetase, enhanced the salt tolerance of both tobacco and Arabidopsis by polyamine synthesis [55], which was verified by the protein interaction of CaM4 with SAMS1 governing ion homeostasis and ethylene synthesis [56].
Phytohormones, as endogenous substances, play significant roles in modulating plant germination, growth, flowering, fruit and other processes, especially at very low concentrations, and their functions are complex and sophisticated [57,58]. The phytohormone strategy used to solve HM poisoning in plants has attracted strong attention [59], including foliar application of exogenous phytohormones and endogenous regulation of auxin, abscisic acid (ABA), ethylene (ETH) and others [60,61,62]. Auxins help plants respond to heavy metal toxicity by regulating their biosynthesis, degradation, signal transduction and transport and play an important role in root development under normal and stress conditions [63]. Auxin plays an important role in root development under normal and stress conditions. In tomato, SI-IAA27 was identified as a pivotal gene that passes real-time information between auxin, ethylene biosynthesis and strigolactone biosynthesis through transcription regulation modules such as SI-IAA27-SI-ERF. B3 and SI-IAA27-SI-NSP1 [64]. Moreover, overexpressing ApHIPP26 inhibited the activity of the ethylene pathway, in which three genes and the key gene in ethylene synthesis, ACS2, were all downregulated, which is consistent with the results showing that inhibiting ethylene synthesis can enhance cadmium tolerance in plants [65,66]. In our study, AtIAA27 (AT4G29080) in auxin and AtERF1 (AT3G23240) in the ethylene pathway were differentially expressed. The ABA signaling pathway consists of several core components (e.g., Snfl-related protein kinase 2, SnRK2s; 2C-type protein phosphatase, PP2Cs; ABA-response element binding factors and ABF), which play an important role in the response to abiotic stress [67]. The transcription factor MYB49 promotes the accumulation of Cd by binding the promoter of HIPP22 to HIPP44, while the accumulation of Cd in Arabidopsis leads to an increase in endogenous ABA content, which induces ABI5 expression and inhibits MYB49 binding to downstream regulatory genes to achieve negative feedback regulation [25]. ABI5 interacts with HAB1/ABI2 by dephosphorylation [68]. In this work, DEGs (HAB1/ABI2) related to the negative regulator PP2C were downregulated, which further activated downstream ABF-binding factors, thereby enhancing ABA signal transduction, which suggested that ABA signal transduction was activated by overexpression of ApHIPP26 and enhanced Cd accumulation. However, this conjecture contradicts some research findings that high ABA levels enable Cd deposition in apoplastic obstacles and inhibit the transport of Cd via apoplastic pathways [69,70]. We believe that whether ABA promotes metal accumulation depends on the metal concentration and plant species, and the mechanism is still complex [71].

4. Materials and Methods

4.1. Plant Culture and Treatments

The materials used in this experiment were A. thaliana, A. paniculata and Nicotiana tabacum Linn. (Huaxi District, Guiyang, China, E106.67°, N26.41°). Tobacco seeds were directly sown in a square box filled with nutrient soil and the seedlings were transplanted after two weeks. Arabidopsis seeds were washed with alcohol and then washed with sterile water 3 times, then were vernalized at 4 °C for 3 days, germinated vertically on a Murashige and Skoog (MS) medium for 7 days. After three weeks of growth in the nutrient soil, six pots of wild-type Arabidopsis and three transgenic lines (four plants per pot) were selected and divided into two groups. The Hoagland medium with or without 2.5 mmol/L CdCl2 was used to irrigate for 14 days. The seeds of Arabidopsis were washed in the same way and root length and germination rate were measured on media with or without 100 μm CdCl2 and 150 μm CdCl2 on a 1/2 Murashige and Skoog (1/2MS) medium. Three biological replicates were set for each group. After three weeks in Hoagland solution, A. paniculata seedlings were treated with 0.25 mM CdCl2 for seven days. The artificial climate incubator strictly implements the light and humidity control system with specific indicators referring to a published article [66].

4.2. Phylogenetic Analysis

Using the protein sequence translated from the coding sequence of ApHIPP26 as a query condition, the homologous ApHIPP26 protein of A. thaliana, Eutrema salsugineum (Pall.) Al-Shehbaz, Brassica rapa L., Capsella rubella Reut. ex Boiss., Raphanus sativus L., Camelina sativa (L.) Crantz, Brassica oleracea L., Tarenaya hassleriana (Chodat) H.E. Moore, Salvia splendens Sellow ex Roem., Phtheirospermum japonicum (Thunb.) Kanitz, and Solanum lycopersicum L. MEGA7.0 was used to analyse the phylogenetic relationships of HIPP26 proteins among these 11 species. DNAMAN was used to analyse the multiple sequence alignment of HIPP26 protein among these 11 species.

4.3. Subcellular Localization

Primers were designed according to the CDs sequence of the HIPP26 gene and the pROKII vector with restriction sites. The pROKII-ApHIPP26-GFP vector was constructed by homologous recombination and transformed into Agrobacterium GV3101 by the freeze-thaw method. The prepared bacterial liquid was injected into the tobacco leaves, and the tobacco seedlings were put into an artificial climate incubator for dark treatment and cultured for two days. The blade was cut into 1 cm2 pieces to make temporary slides, which were observed and photographed with a laser confocal microscope (Zeiss, LSM510 Meta, Carl Zeiss). The pROKII empty vector was used as the control.

4.4. RNA Isolation and qRT–PCR

Total RNA extraction was performed with an RN38-EASYspin Plus Kit (Aidlab, Hangzhou, China) and RT Master Mix for qPCR II Kit (MedChemExpress, Shanghai, China) for reverse transcription. The BIOER FQD-48A system (BIOER, Hangzhou, China) was utilized for RT-qPCR. The gene sequences of primers used are shown in the attached table, and the reference gene is Actin 1. The 2−ΔΔCT method, which was reported previously [72], was employed to estimate gene expression.

4.5. Construction of the Expression Vector and Genetic Transformation of A. thaliana

The construction of the pB121-HIPP26 vector was based on a published method [28]. A. thaliana growing to bloom was used for genetic transformation mediated by Agrobacterium GV3101 [29]. Screening of positive plants was carried out by 1/2 MS medium containing 50 mg/L kanamycin, and T3 seedlings were obtained for subsequent study.

4.6. Phenotypic Analysis and Determination of Physiological Indices

The antioxidant enzymes in aboveground parts were determined, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and the levels of malondialdehyde (MDA), proline (Pro), and chlorophyll. All substances were quantified using a detection kit (Solarbio, Beijing, China) that contained the details of the experimental procedure.

4.7. Determination of Cd Content

Cd accumulation in the shoots and roots of the transgenic lines (OE2, OE5 and OE9) and wild type (WT) was determined. It should be noted that the roots of the plant should be sufficiently cleaned to ensure that Cd in solution was removed. The plant samples were dried and ground into powder, with specific operational details referring to published papers [73], and then concentration measurements were performed using ICP-MS [74].

4.8. RNA-Seq Analysis

We selected aboveground tissues of transgenic plants and WT plants for transcriptome sequencing. The two groups were treated with different concentrations of Cd (0 mM CdCl2, 2.5 mM CdCl2). A NanoDrop 2000 spectrophotometer, which is a professional platform to quantify the samples, was initially used to quantify the RNA concentration. Then, an Agilent 2100/4200 was used to interpret the RNA sample quality and accurately quantify the concentration. When the construction of the library was finished, Qubit 3.0 software was available and used for initial quantification. The Illumina NovaSeq 6000 possessed advanced sequencing technology and was used for transcriptome sequencing in this study after library inspection was qualified. The raw sequence data are available in NCBI-SRA (https://www.ncbi.nlm.nih.gov/sra (accessed on 23 August 2023)) under the accession number PRJNA947359.4.9. Validation was conducted using real-time quantitative PCR (RT-qPCR).

4.9. Statistical Analysis

Regression analysis was performed using SPSS V25, which is suitable for analysis of variance between groups. The data shown in the article and every treatment group were subjected to the experiment 3 times.

4.10. Primers

All the primers used in this study are listed in Table S1.

5. Conclusions

In this work, we identified the Cd-induced gene HIPP26 from A. paniculata and transformed it into A. thaliana. ApHIPP26 overexpression plants displayed Cd tolerance and accumulation in physiological and biochemical performances through enhancing the activity of antioxidant enzymes and modifying the expression of genes involved in the plant hormone pathways. These findings will help reveal the mechanism of Cd tolerance in hyperaccumulators, which may contribute to phytoremediation and even provide useful candidate molecular markers for breeding low-cadmium crops. As we all know, heavy metals have a strong toxic effect on organisms, seriously threatening our health, so reducing the pollution and accumulation of heavy metals is extremely important. In future studies, we may be able to find more hyperaccumulators to achieve the remediation of soil and water. It is also possible to transfer the key genes in the hyperaccumulators or wild relatives of cultivated species [75,76] into crops through transgenic technology to cultivate crops that are resistant to heavy metals and can be enriched in non-edible parts.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242015052/s1.

Author Contributions

Conceptualization, H.W. and B.Z.; methodology, Y.H.; software, B.P.; valida- tion, H.W. and B.Z.; investigation, L.Y.; data curation, J.Y.; writing—original draft preparation, L.Z. and L.Y.; writing—review and editing, L.Z.; visualization, L.Z.; supervision, X.D.; project administration, L.G.; funding acquisition, H.W. and B.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Precursor Projects of Guizhou Province for Biological Breeding Supporting by Science and Technology in 2022 (QKHZC2022ZD031), National Natural Science Foundation of China (grant no. 32260460).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. An, J.-P.; Wang, X.-N.; Yao, J.-F.; Ren, Y.-R.; You, C.-X.; Wang, X.-F.; Hao, Y.-J. Apple MdMYC2 Reduces Aluminum Stress Tolerance by Directly Regulating MdERF3 Gene. Plant Soil 2017, 418, 255–266. [Google Scholar] [CrossRef]
  2. Khoudi, H. Significance of Vacuolar Proton Pumps and Metal/H+ Antiporters in Plant Heavy Metal Tolerance. Physiol. Plant. 2021, 173, 384–393. [Google Scholar] [CrossRef] [PubMed]
  3. Kaya, C.; Higgs, D.; Ashraf, M.; Alyemeni, M.N.; Ahmad, P. Integrative Roles of Nitric Oxide and Hydrogen Sulfide in Melatonin-induced Tolerance of Pepper (Capsicum annuum L.) Plants to Iron Deficiency and Salt Stress Alone or in Combination. Physiol. Plant. 2019, 168, 256–277. [Google Scholar] [CrossRef]
  4. Maharajan, T.; Chellasamy, G.; Tp, A.K.; Ceasar, S.A.; Yun, K. The Role of Metal Transporters in Phytoremediation: A Closer Look at Arabidopsis. Chemosphere 2023, 310, 136881. [Google Scholar] [CrossRef]
  5. Akindele, E.O.; Omisakin, O.D.; Oni, O.A.; Aliu, O.O.; Omoniyi, G.E.; Akinpelu, O.T. Heavy metal toxicity in the water column and benthic sediments of a degraded tropical stream. Ecotoxicol. Environ. Saf. 2020, 190, 110153. [Google Scholar] [CrossRef]
  6. Ali, B.; Tao, Q.; Zhou, Y.; Gill, R.A.; Ali, S.; Rafiq, M.T.; Xu, L.; Zhou, W. 5-Aminolevolinic Acid Mitigates the Cadmium-Induced Changes in Brassica napus as Revealed by the Biochemical and Ultra-Structural Evaluation of Roots. Ecotoxicol. Environ. Saf. 2013, 92, 271–280. [Google Scholar] [CrossRef] [PubMed]
  7. Ali, B.; Qian, P.; Jin, R.; Ali, S.; Khan, M.; Aziz, R.; Tian, T.; Zhou, W. Physiological and Ultra-Structural Changes in Brassica napus Seedlings Induced by Cadmium Stress. Biol. Plant. 2014, 58, 131–138. [Google Scholar] [CrossRef]
  8. Ali, B.; Gill, R.A.; Yang, S.; Gill, M.B.; Ali, S.; Rafiq, M.T.; Zhou, W. Hydrogen Sulfide Alleviates Cadmium-Induced Morpho-Physiological and Ultrastructural Changes in Brassica napus. Ecotoxicol. Environ. Saf. 2014, 110, 197–207. [Google Scholar] [CrossRef]
  9. Zhang, W.; Song, J.; Yue, S.; Duan, K.; Yang, H. MhMAPK4 from Malus Hupehensis Rehd. Decreases Cell Death in Tobacco Roots by Controlling Cd2+ Uptake. Ecotoxicol. Environ. Saf. 2019, 168, 230–240. [Google Scholar] [CrossRef]
  10. Ankit; Saha, L.; Kumar, V.; Tiwari, J.; Sweta; Rawat, S.; Singh, J.; Bauddh, K. Electronic Waste and Their Leachates Impact on Human Health and Environment: Global Ecological Threat and Management. Environ. Technol. Innov. 2021, 24, 102049. [Google Scholar] [CrossRef]
  11. Liao, R.; Ratié, G.; Shi, Z.; Šípková, A.; Vaňková, Z.; Chrastný, V.; Zhang, J.; Komárek, M. Cadmium Isotope Systematics for Source Apportionment in an Urban–Rural Region. Appl. Geochem. 2022, 137, 105196. [Google Scholar] [CrossRef]
  12. Nocito, F.F.; Lancilli, C.; Dendena, B.; Lucchini, G.; Sacchi, G.A. Cadmium Retention in Rice Roots Is Influenced by Cadmium Availability, Chelation and Translocation: Cadmium Retention in Rice Roots. Plant Cell Environ. 2011, 34, 994–1008. [Google Scholar] [CrossRef]
  13. Yamaji, N.; Sasaki, A.; Xia, J.X.; Yokosho, K.; Ma, J.F. A Node-Based Switch for Preferential Distribution of Manganese in Rice. Nat. Commun. 2013, 4, 2442. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, R.; Sang, P.; Guo, Y.; Jin, P.; Cheng, Y.; Yu, H.; Xie, Y.; Yao, W.; Qian, H. Cadmium in Food: Source, Distribution and Removal. Food Chem. 2023, 405, 134666. [Google Scholar] [CrossRef] [PubMed]
  15. Castaings, L.; Caquot, A.; Loubet, S.; Curie, C. The High-Affinity Metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision. Sci. Rep. 2016, 6, 37222. [Google Scholar] [CrossRef] [PubMed]
  16. Dykema, P.E.; Sipes, P.R.; Marie, A.; Biermann, B.J.; Crowell, D.N.; Randall, S.K. A New Class of Proteins Capable of Binding Transition Metals. Plant Mol. Biol. 1999, 41, 139–150. [Google Scholar] [CrossRef]
  17. Gao, W.; Xiao, S.; Li, H.; Tsao, S.; Chye, M. Arabidopsis thaliana acyl-CoA-binding Protein ACBP2 Interacts with Heavy-metal-binding Farnesylated Protein AtFP6. New Phytol. 2009, 181, 89–102. [Google Scholar] [CrossRef] [PubMed]
  18. Parasyri, A.; Barth, O.; Zschiesche, W.; Humbeck, K. The Barley Heavy Metal Associated Isoprenylated Plant Protein HvFP1 Is Involved in a Crosstalk between the Leaf Development and Abscisic Acid-Related Drought Stress Responses. Plants 2022, 11, 2851. [Google Scholar] [CrossRef]
  19. Wei, Y.; Peng, X.; Wang, X.; Wang, C. The Heavy Metal-Associated Isoprenylated Plant Protein (HIPP) Gene Family Plays a Crucial Role in Cadmium Resistance and Accumulation in the Tea Plant (Camellia sinensis L.). Ecotoxicol. Environ. Saf. 2023, 260, 115077. [Google Scholar] [CrossRef] [PubMed]
  20. Khan, I.U.; Rono, J.K.; Zhang, B.Q.; Liu, X.S.; Wang, M.Q.; Wang, L.L.; Wu, X.C.; Chen, X.; Cao, H.W.; Yang, Z.M. Identification of Novel Rice (Oryza Sativa) HPP and HIPP Genes Tolerant to Heavy Metal Toxicity. Ecotoxicol. Environ. Saf. 2019, 175, 8–18. [Google Scholar] [CrossRef]
  21. Ye, X.; Liu, C.; Yan, H.; Wan, Y.; Wu, Q.; Wu, X.; Zhao, G.; Zou, L.; Xiang, D. Genome-Wide Identification and Transcriptome Analysis of the Heavy Metal-Associated (HMA) Gene Family in Tartary Buckwheat and Their Regulatory Roles under Cadmium Stress. Gene 2022, 847, 146884. [Google Scholar] [CrossRef] [PubMed]
  22. Suzuki, N.; Yamaguchi, Y.; Koizumi, N.; Sano, H. Functional Characterization of a Heavy Metal Binding Protein CdI19 from Arabidopsis. Plant J. 2002, 32, 165–173. [Google Scholar] [CrossRef]
  23. Zhao, Y.N.; Wang, M.Q.; Li, C.; Cao, H.W.; Rono, J.K.; Yang, Z.M. The Metallochaperone OsHIPP56 Gene Is Required for Cadmium Detoxification in Rice Crops. Environ. Exp. Bot. 2022, 193, 104680. [Google Scholar] [CrossRef]
  24. De Abreu-Neto, J.B.; Turchetto-Zolet, A.C.; De Oliveira, L.F.V.; Bodanese Zanettini, M.H.; Margis-Pinheiro, M. Heavy Metal-Associated Isoprenylated Plant Protein (HIPP): Characterization of a Family of Proteins Exclusive to Plants. FEBS J. 2013, 280, 1604–1616. [Google Scholar] [CrossRef] [PubMed]
  25. Zhang, P.; Wang, R.; Ju, Q.; Li, W.; Tran, L.-S.P.; Xu, J. The R2R3-MYB Transcription Factor MYB49 Regulates Cadmium Accu mulation. Plant Physiol. 2019, 180, 529–542. [Google Scholar] [CrossRef]
  26. Tang, Y.-T.; Qiu, R.-L.; Zeng, X.-W.; Ying, R.-R.; Yu, F.-M.; Zhou, X.-Y. Lead, Zinc, Cadmium Hyperaccumulation and Growth Stimulation in Arabis paniculata Franch. Environ. Exp. Bot. 2009, 66, 126–134. [Google Scholar] [CrossRef]
  27. Diao, F.; Dang, Z.; Cui, X.; Xu, J.; Jia, B.; Ding, S.; Zhang, Z.; Guo, W. Transcriptomic Analysis Revealed Distinctive Modulations of Arbuscular Mycorrhizal Fungi Inoculation in Halophyte suaeda Salsa under Moderate Salt Conditions. Environ. Exp. Bot. 2021, 183, 104337. [Google Scholar] [CrossRef]
  28. Renu, K.; Chakraborty, R.; Myakala, H.; Koti, R.; Famurewa, A.C.; Madhyastha, H.; Vellingiri, B.; George, A.; Valsala Gopalakrishnan, A. Molecular Mechanism of Heavy Metals (Lead, Chromium, Arsenic, Mercury, Nickel and Cadmium)—Induced Hepatotoxicity—A Review. Chemosphere 2021, 271, 129735. [Google Scholar] [CrossRef]
  29. Gerhardt, K.E.; Gerwing, P.D.; Greenberg, B.M. Opinion: Taking Phytoremediation from Proven Technology to Accepted Practice. Plant Sci. 2017, 256, 170–185. [Google Scholar] [CrossRef]
  30. Wuana, R.A.; Okieimen, F.E. Heavy Metals in Contaminated Soils: A Review of Sources, Chemistry, Risks and Best Available Strategies for Remediation. ISRN Ecol. 2011, 2011, 402647. [Google Scholar] [CrossRef]
  31. DalCorso, G.; Fasani, E.; Manara, A.; Visioli, G.; Furini, A. Heavy Metal Pollutions: State of the Art and Innovation in Phytore mediation. IJMS 2019, 20, 3412. [Google Scholar] [CrossRef]
  32. Ali, H.; Khan, E.; Sajad, M.A. Phytoremediation of Heavy Metals—Concepts and Applications. Chemosphere 2013, 91, 869–881. [Google Scholar] [CrossRef]
  33. Sivarajasekar, N.; Baskar, R. Biosorption of Basic Violet 10 onto Activated Gossypium hirsutum Seeds: Batch and Fixed-Bed Column Studies. Chin. J. Chem. Eng. 2015, 23, 1610–1619. [Google Scholar] [CrossRef]
  34. Parmar, S.; Singh, V. Phytoremediation approaches for heavy metal pollution: A review. J. Plant Sci. Res. 2015, 2, 135–139. [Google Scholar]
  35. Li, S.; Zhuo, R.; Yu, M.; Lin, X.; Xu, J.; Qiu, W.; Li, H.; Han, X. A Novel Gene SpCTP3 from the Hyperaccumulator Sedum plumbizincicola Redistributes Cadmium and Increases Its Accumulation in Transgenic Populus × Canescens. Front. Plant Sci. 2023, 14, 1111789. [Google Scholar] [CrossRef] [PubMed]
  36. Liu, H.; Zhao, H.; Wu, L.; Liu, A.; Zhao, F.; Xu, W. Heavy Metal ATPase 3 (HMA3) Confers Cadmium Hypertolerance on the Cadmium/Zinc Hyperaccumulator Sedum plumbizincicola. New Phytol. 2017, 215, 687–698. [Google Scholar] [CrossRef]
  37. Lin, J.; Gao, X.; Zhao, J.; Zhang, J.; Chen, S.; Lu, L. Plant Cadmium Resistance 2 (SaPCR2) Facilitates Cadmium Efflux in the Roots of Hyperaccumulator Sedum alfredii Hance. Front. Plant Sci. 2020, 11, 568887. [Google Scholar] [CrossRef] [PubMed]
  38. Fasani, E.; DalCorso, G.; Zorzi, G.; Agrimonti, C.; Fragni, R.; Visioli, G.; Furini, A. Overexpression of ZNT1 and NRAMP4 from the Ni Hyperaccumulator Noccaea Caerulescens Population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni Accumulation. IJMS 2021, 22, 11896. [Google Scholar] [CrossRef]
  39. Feng, S.; Tan, J.; Zhang, Y.; Liang, S.; Xiang, S.; Wang, H.; Chai, T. Isolation and Characterization of a Novel Cadmium-Regu lated Yellow Stripe-Like Transporter (SnYSL3) in Solanum nigrum. Plant Cell Rep. 2017, 36, 281–296. [Google Scholar] [CrossRef]
  40. Chen, H.; Zhang, C.; Guo, H.; Hu, Y.; He, Y.; Jiang, D. Overexpression of a Miscanthus Sacchariflorus Yellow Stripe-like Trans porter MsYSL1 Enhances Resistance of Arabidopsis to Cadmium by Mediating Metal Ion Reallocation. Plant Growth Regul. 2018, 85, 101–111. [Google Scholar] [CrossRef]
  41. Liu, Z.; Zhou, L.; Gan, C.; Hu, L.; Pang, B.; Zuo, D.; Wang, G.; Wang, H.; Liu, Y. Transcriptomic Analysis Reveals Key Genes and Pathways Corresponding to Cd and Pb in the Hyperaccumulator Arabis paniculata. Ecotoxicol. Environ. Saf. 2023, 254, 114757. [Google Scholar] [CrossRef] [PubMed]
  42. Parveen, N.; Kandhol, N.; Sharma, S.; Singh, V.P.; Chauhan, D.K.; Ludwig-Müller, J.; Corpas, F.J.; Tripathi, D.K. Auxin Cross talk with Reactive Oxygen and Nitrogen Species in Plant Development and Abiotic Stress. Plant Cell Physiol. 2023, 63, 1814–1825. [Google Scholar] [CrossRef] [PubMed]
  43. Mansoor, S.; Ali Wani, O.; Lone, J.K.; Manhas, S.; Kour, N.; Alam, P.; Ahmad, A.; Ahmad, P. Reactive Oxygen Species in Plants: From Source to Sink. Antioxidants 2022, 11, 225. [Google Scholar] [CrossRef] [PubMed]
  44. Sedaghat, M.; Tahmasebi-Sarvestani, Z.; Emam, Y.; Mokhtassi-Bidgoli, A. Physiological and Antioxidant Responses of Winter Wheat Cultivars to Strigolactone and Salicylic Acid in Drought. Plant Physiol. Biochem. 2017, 119, 59–69. [Google Scholar] [CrossRef]
  45. Cui, D.; Yin, Y.; Wang, J.; Wang, Z.; Ding, H.; Ma, R.; Jiao, Z. Research on the Physio-Biochemical Mechanism of Non-Thermal Plasma-Regulated Seed Germination and Early Seedling Development in Arabidopsis. Front. Plant Sci. 2019, 10, 1322. [Google Scholar] [CrossRef]
  46. Liu, Z.; He, X.; Chen, W.; Yuan, F.; Yan, K.; Tao, D. Accumulation and Tolerance Characteristics of Cadmium in a Potential Hyperaccumulator—Lonicera Japonica Thunb. J. Hazard. Mater. 2009, 169, 170–175. [Google Scholar] [CrossRef]
  47. Lu, Z.; Chen, S.; Han, X.; Zhang, J.; Qiao, G.; Jiang, Y.; Zhuo, R.; Qiu, W. A Single Amino Acid Change in Nramp6 from Sedum alfredii Hance Affects Cadmium Accumulation. IJMS 2020, 21, 3169. [Google Scholar] [CrossRef]
  48. Jia, H.; Yin, Z.; Xuan, D.; Lian, W.; Han, D.; Zhu, Z.; Li, C.; Li, C.; Song, Z. Mutation of NtNRAMP3 Improves Cadmium Tolerance and Its Accumulation in Tobacco Leaves by Regulating the Subcellular Distribution of Cadmium. J. Hazard. Mater. 2022, 432, 128701. [Google Scholar] [CrossRef]
  49. Wang, D.; Repo, E.; He, F.; Zhang, X.; Xiang, H.; Yang, W.; Min, X.; Zhao, F. Dual Functional Sites Strategies toward Enhanced Heavy Metal Remediation: Interlayer Expanded Mg-Al Layered Double Hydroxide by Intercalation with L-Cysteine. J. Hazard. Mater. 2022, 439, 129693. [Google Scholar] [CrossRef]
  50. Tao, Y.; Wu, Q.; Huang, J.; Fang Shen, R.; Zhu, X.F. The Upstream Regulation of the Root Cell Wall When Arabidopsis Thaliana in Response to Toxic Metal Ions Focusing on Al. Plant Signal. Behav. 2023, 18, 2178085. [Google Scholar] [CrossRef]
  51. Kumar, S.; Yadav, A.; Verma, R.; Dubey, A.K.; Narayan, S.; Pandey, A.; Sahu, A.; Srivastava, S.; Sanyal, I. Metallothionein (MT1): A Molecular Stress Marker in Chickpea Enhances Drought and Heavy Metal Stress Adaptive Efficacy in Transgenic Plants. Environ. Exp. Bot. 2022, 199, 104871. [Google Scholar] [CrossRef]
  52. Li, R.; Yang, Y.; Cao, H.; Peng, X.; Yu, Q.; He, L.; Chen, J.; Xiang, L.; Liu, W. Heterologous Expression of the Tobacco Metal lothionein Gene NtMT2F Confers Enhanced Tolerance to Cd Stress in Escherichia Coli and Arabidopsis thaliana. Plant Physiol. Biochem. 2023, 195, 247–255. [Google Scholar] [CrossRef] [PubMed]
  53. Kim, Y.-O.; Gwon, Y.; Kim, J. Exogenous Cysteine Improves Mercury Uptake and Tolerance in Arabidopsis by Regulating the Expression of Heavy Metal Chelators and Antioxidative Enzymes. Front. Plant Sci. 2022, 13, 898247. [Google Scholar] [CrossRef] [PubMed]
  54. Yuan, Y.; Song, T.; Yu, J.; Zhang, W.; Hou, X.; Kong Ling, Z.; Cui, G. Genome-Wide Investigation of the Cysteine Synthase Gene Family Shows That Overexpression of CSase Confers Alkali Tolerance to Alfalfa (Medicago sativa L.). Front. Plant Sci. 2022, 12, 792862. [Google Scholar] [CrossRef] [PubMed]
  55. Gong, B.; Li, X.; VandenLangenberg, K.M.; Wen, D.; Sun, S.; Wei, M.; Li, Y.; Yang, F.; Shi, Q.; Wang, X. Overexpression of S-Adenosyl- l -Methionine Synthetase Increased Tomato Tolerance to Alkali Stress through Polyamine Metabolism. Plant Biotechnol. J. 2014, 12, 694–708. [Google Scholar] [CrossRef]
  56. Yang, S.; Wang, J.; Tang, Z.; Li, Y.; Zhang, J.; Guo, F.; Meng, J.; Cui, F.; Li, X.; Wan, S. Calcium/Calmodulin Modulates Salt Responses by Binding a Novel Interacting Protein SAMS1 in Peanut (Arachis hypogaea L.). Crop J. 2023, 11, 21–32. [Google Scholar] [CrossRef]
  57. Jiang, K.; Asami, T. Chemical Regulators of Plant Hormones and Their Applications in Basic Research and Agriculture. Biosci. Biotechnol. Biochem. 2018, 82, 1265–1300. [Google Scholar] [CrossRef] [PubMed]
  58. Wani, S.H.; Kumar, V.; Shriram, V.; Sah, S.K. Phytohormones and Their Metabolic Engineering for Abiotic Stress Tolerance in Crop Plants. Crop J. 2016, 4, 162–176. [Google Scholar] [CrossRef]
  59. Sytar, O.; Ghosh, S.; Malinska, H.; Zivcak, M.; Brestic, M. Physiological and Molecular Mechanisms of Metal Accumulation in Hyperaccumulator Plants. Physiol. Plant. 2020, 173, 148–166. [Google Scholar] [CrossRef]
  60. Bücker-Neto, L.; Paiva, A.L.S.; Machado, R.D.; Arenhart, R.A.; Margis-Pinheiro, M. Interactions between Plant Hormones and Heavy Metals Responses. Genet. Mol. Biol. 2017, 40, 373–386. [Google Scholar] [CrossRef]
  61. Huang, T.-L.; Nguyen, Q.T.T.; Fu, S.-F.; Lin, C.-Y.; Chen, Y.-C.; Huang, H.-J. Transcriptomic Changes and Signalling Pathways Induced by Arsenic Stress in Rice Roots. Plant Mol. Biol. 2012, 80, 587–608. [Google Scholar] [CrossRef] [PubMed]
  62. Chen, L.; Yang, H.; Fang, Y.; Guo, W.; Chen, H.; Zhang, X.; Dai, W.; Chen, S.; Hao, Q.; Yuan, S.; et al. Overexpression of GmMYB14 Improves High-density Yield and Drought Tolerance of Soybean through Regulating Plant Architecture Mediated by the Brassinosteroid Pathway. Plant Biotechnol. J. 2021, 19, 702–716. [Google Scholar] [CrossRef] [PubMed]
  63. Ejaz, U.; Khan, S.M.; Khalid, N.; Ahmad, Z.; Jehangir, S.; Fatima Rizvi, Z.; Lho, L.H.; Han, H.; Raposo, A. Detoxifying the Heavy Metals: A Multipronged Study of Tolerance Strategies against Heavy Metals Toxicity in Plants. Front. Plant Sci. 2023, 14, 1154571. [Google Scholar] [CrossRef] [PubMed]
  64. Guillotin, B.; Etemadi, M.; Audran, C.; Bouzayen, M.; Bécard, G.; Combier, J. Sl-IAA27 Regulates Strigolactone Biosynthesis and Mycorrhization in Tomato (Var. MicroTom). New Phytol. 2017, 213, 1124–1132. [Google Scholar] [CrossRef] [PubMed]
  65. Zuo, D.; Hu, M.; Zhou, W.; Lei, F.; Zhao, J.; Gu, L. EcAGL Enhances Cadmium Tolerance in Transgenic Arabidopsis Thaliana through Inhibits Cadmium Transport and Ethylene Synthesis Pathway. Plant Physiol. Biochem. 2023, 201, 107900. [Google Scholar] [CrossRef] [PubMed]
  66. Wang, H.; Zuo, D.; Zhu, B.; Du, X.; Gu, L. TaMYC8 Regulates TaERF6 and Inhibits Ethylene Synthesis to Confer Cd Tolerance in Wheat. Environ. Exp. Bot. 2022, 198, 104854. [Google Scholar] [CrossRef]
  67. Ng, L.M.; Melcher, K.; Teh, B.T.; Xu, H.E. Abscisic Acid Perception and Signaling: Structural Mechanisms and Applications. Acta Pharmacol. Sin. 2014, 35, 567–584. [Google Scholar] [CrossRef] [PubMed]
  68. Skubacz, A.; Daszkowska-Golec, A.; Szarejko, I. The Role and Regulation of ABI5 (ABA-Insensitive 5) in Plant Development, Abiotic Stress Responses and Phytohormone Crosstalk. Front. Plant Sci. 2016, 7, 1884. [Google Scholar] [CrossRef]
  69. Meng, L.; Yang, Y.; Ma, Z.; Jiang, J.; Zhang, X.; Chen, Z.; Cui, G.; Yin, X. Integrated Physiological, Transcriptomic and Metab olomic Analysis of the Response of Trifolium pratense L. to Pb Toxicity. J. Hazard. Mater. 2022, 436, 129128. [Google Scholar] [CrossRef]
  70. Zhang, W.; Wang, Z.; Song, J.; Yue, S.; Yang, H. Cd2+ Uptake Inhibited by MhNCED3 from Malus Hupehensis Alleviates Cd-Induced Cell Death. Environ. Exp. Bot. 2019, 166, 103802. [Google Scholar] [CrossRef]
  71. Lu, Q.; Chen, S.; Li, Y.; Zheng, F.; He, B.; Gu, M. Exogenous Abscisic Acid (ABA) Promotes Cadmium (Cd) Accumulation in Sedum Alfredii Hance by Regulating the Expression of Cd Stress Response Genes. Environ. Sci. Pollut. Res. 2020, 27, 8719–8731. [Google Scholar] [CrossRef] [PubMed]
  72. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  73. Du, X.; Fang, L.; Li, J.; Chen, T.; Cheng, Z.; Zhu, B.; Gu, L.; Wang, H. The TabHLH094–TaMYC8 Complex Mediates the Cad mium Response in Wheat. Mol. Breed. 2023, 43, 57. [Google Scholar] [CrossRef] [PubMed]
  74. Kamar, V.; Dağalp, R.; Taştekin, M. Determination of Heavy Metals in Almonds and Mistletoe as a Parasite Growing on the Almond Tree Using ICP-OES or ICP-MS. Biol. Trace Elem. Res. 2018, 185, 226–235. [Google Scholar] [CrossRef]
  75. Perrino, E.V.; Brunetti, G.; Farrag, K. Plant communities of multi-metal contaminated soils: A case study in National Park of Alta Murgia (Apulia Region—Southern Italy). Int. J. Phytoremediat. 2014, 16, 871–888. [Google Scholar] [CrossRef]
  76. Perrino, E.V.; Wagensommer, R.P.; Medagli, P. The genus Aegilops L. (Poaceae) in Italy: Taxonomy, geographical distribution, ecology, vulnerability and conservation. Syst. Biodivers. 2014, 12, 331–349. [Google Scholar] [CrossRef]
Ijms 24 15052 g001
Figure 1. Phylogenetic relationship (a) and multiple sequence alignment (b) between ApHIPP26 and other plant homologous proteins.
Figure 1. Phylogenetic relationship (a) and multiple sequence alignment (b) between ApHIPP26 and other plant homologous proteins.
Ijms 24 15052 g001
Ijms 24 15052 g002
Figure 2. ApHIPP26 is localized in the nucleus. The 35S:ApHIPP26-GFP fusion protein was transiently expressed in tobacco. Scale bar = 50 μm.
Figure 2. ApHIPP26 is localized in the nucleus. The 35S:ApHIPP26-GFP fusion protein was transiently expressed in tobacco. Scale bar = 50 μm.
Ijms 24 15052 g002
Ijms 24 15052 g003
Figure 3. Expression patterns of the ApHIPP26 gene. (a) Relative expression of ApHIPP26 under cadmium (Cd) stress in Arabis paniculata. (b) Relative ApHIPP26 expression in transgenic Arabidopsis overexpressing the ApHIPP26 gene. Different letters indicate a statistically significant difference at p < 0.01.
Figure 3. Expression patterns of the ApHIPP26 gene. (a) Relative expression of ApHIPP26 under cadmium (Cd) stress in Arabis paniculata. (b) Relative ApHIPP26 expression in transgenic Arabidopsis overexpressing the ApHIPP26 gene. Different letters indicate a statistically significant difference at p < 0.01.
Ijms 24 15052 g003
Ijms 24 15052 g004
Figure 4. The growth state of Arabidopsis overexpressing the ApHIPP26 gene under cadmium (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 without or containing 100 µM and 150 µM Cd. Scale bar = 1.5 cm. (c) Phenotypes morphology of wild-type and transgenic Arabidopsis treated with 0 or 2.5 mM Cd for 14 days. Scale bar = 5 cm.
Figure 4. The growth state of Arabidopsis overexpressing the ApHIPP26 gene under cadmium (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 without or containing 100 µM and 150 µM Cd. Scale bar = 1.5 cm. (c) Phenotypes morphology of wild-type and transgenic Arabidopsis treated with 0 or 2.5 mM Cd for 14 days. Scale bar = 5 cm.
Ijms 24 15052 g004
Ijms 24 15052 g005
Figure 5. Detection of root length, percentage of germination, and fresh weight of WT and transgenic Arabidopsis overexpressing the ApHIPP26 gene under cadmium (Cd) stress. (a) Root length. (b) percentage of germination. (c) Fresh weight. Each line after 14 days of growth in media with or without 100 μM Cd. Different letters indicate a statistically significant difference at p < 0.01.
Figure 5. Detection of root length, percentage of germination, and fresh weight of WT and transgenic Arabidopsis overexpressing the ApHIPP26 gene under cadmium (Cd) stress. (a) Root length. (b) percentage of germination. (c) Fresh weight. Each line after 14 days of growth in media with or without 100 μM Cd. Different letters indicate a statistically significant difference at p < 0.01.
Ijms 24 15052 g005
Ijms 24 15052 g006
Figure 6. Physiological indices of plants expressing ApHIPP26 under control and cadmium (Cd) stress conditions. (a) Malondialdehyde (MDA). (b) Proline (Pro). (c) Peroxidase (POD) activity. (d) Ascorbate peroxidase (APX) activity. (e) Catalase (CAT). (f) Superoxide dismutase (SOD). (g) Chlorophyll content. Different letters indicate a statistically significant difference at p < 0.01.
Figure 6. Physiological indices of plants expressing ApHIPP26 under control and cadmium (Cd) stress conditions. (a) Malondialdehyde (MDA). (b) Proline (Pro). (c) Peroxidase (POD) activity. (d) Ascorbate peroxidase (APX) activity. (e) Catalase (CAT). (f) Superoxide dismutase (SOD). (g) Chlorophyll content. Different letters indicate a statistically significant difference at p < 0.01.
Ijms 24 15052 g006
Ijms 24 15052 g007aIjms 24 15052 g007b
Figure 7. (a) Cadmium content in roots and buds of wild-type (WT) and transgenic Arabidopsis after 14 days of 2.5 mM Cd treatment. Relative expression levels of (b–i) heavy metal transporters in WT and transgenic lines under 0 mM or 2.5 mM Cd stress. Different letters indicate a statistically significant difference at p < 0.01.
Figure 7. (a) Cadmium content in roots and buds of wild-type (WT) and transgenic Arabidopsis after 14 days of 2.5 mM Cd treatment. Relative expression levels of (b–i) heavy metal transporters in WT and transgenic lines under 0 mM or 2.5 mM Cd stress. Different letters indicate a statistically significant difference at p < 0.01.
Ijms 24 15052 g007aIjms 24 15052 g007b
Ijms 24 15052 g008
Figure 8. Differentially expressed genes (DEGs) in the shoots of wild-type (WT) plants and the transgenic line OE5 overexpressing ApHIPP26 under cadmium (Cd) stress identified by transcriptome analysis. (a) The number of DEGs. (b) DEGs enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the shoots of WT and OE5 plants affected by Cd stress. (c) Gene Ontology (GO) assignment of DEGs in the shoots of WT and OE5 plants affected by Cd stress. (d) Expression of DEGs related to Plant hormone signal transduction pathway.
Figure 8. Differentially expressed genes (DEGs) in the shoots of wild-type (WT) plants and the transgenic line OE5 overexpressing ApHIPP26 under cadmium (Cd) stress identified by transcriptome analysis. (a) The number of DEGs. (b) DEGs enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the shoots of WT and OE5 plants affected by Cd stress. (c) Gene Ontology (GO) assignment of DEGs in the shoots of WT and OE5 plants affected by Cd stress. (d) Expression of DEGs related to Plant hormone signal transduction pathway.
Ijms 24 15052 g008
Ijms 24 15052 g009aIjms 24 15052 g009b
Figure 9. (a–h) RNA-seq gene validation was performed using qPC. According to the LSD test, the average FPKM value and relative expression level were shown. Different letters indicate a statistically significant difference at p < 0.01.
Figure 9. (a–h) RNA-seq gene validation was performed using qPC. According to the LSD test, the average FPKM value and relative expression level were shown. Different letters indicate a statistically significant difference at p < 0.01.
Ijms 24 15052 g009aIjms 24 15052 g009b
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhou, L.; Ye, L.; Pang, B.; Hou, Y.; Yu, J.; Du, X.; Gu, L.; Wang, H.; Zhu, B. Overexpression of ApHIPP26 from the Hyperaccumulator Arabis paniculata Confers Enhanced Cadmium Tolerance and Accumulation to Arabidopsis thaliana. Int. J. Mol. Sci. 2023, 24, 15052. https://doi.org/10.3390/ijms242015052

AMA Style

Zhou L, Ye L, Pang B, Hou Y, Yu J, Du X, Gu L, Wang H, Zhu B. Overexpression of ApHIPP26 from the Hyperaccumulator Arabis paniculata Confers Enhanced Cadmium Tolerance and Accumulation to Arabidopsis thaliana. International Journal of Molecular Sciences. 2023; 24(20):15052. https://doi.org/10.3390/ijms242015052

Chicago/Turabian Style

Zhou, Lizhou, Lvlan Ye, Biao Pang, Yunyan Hou, Junxing Yu, Xuye Du, Lei Gu, Hongcheng Wang, and Bin Zhu. 2023. "Overexpression of ApHIPP26 from the Hyperaccumulator Arabis paniculata Confers Enhanced Cadmium Tolerance and Accumulation to Arabidopsis thaliana" International Journal of Molecular Sciences 24, no. 20: 15052. https://doi.org/10.3390/ijms242015052

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop