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Hyperaccumulation in plants is a complex and dynamic biological process. Sedum alfredii, the most studied Cd hyperaccumulator, can accumulate up to 9000 mg kg -1 Cd in its leaves without suffering toxicity. Although several studies have reported the molecular mechanisms of Cd hyperaccumulation, our understanding of the cell-type-specific transcriptional regulation induced by Cd remains limited. In this study, the first full-length transcriptome of S. alfredii was generated using the PacBio Iso-Seq technology. A total of 18,718,513 subreads (39.90 Gb) were obtained, with an average length of 2133 bp. The single-cell RNA sequencing was employed on leaves of S. alfredii grown under Cd stress. A total of 12,616 high-quality single cells were derived from the control and Cd-treatment samples of S. alfredii leaves. Based on cell heterogeneity and the expression profiles of previously reported marker genes, seven cell types with 12 transcriptionally distinct cell clusters were identified, thereby constructing the first single-cell atlas for S. alfredii leaves. Metal transporters such as CAX5, COPT5, ZIP5, YSL7, and MTP1 were up-regulated in different cell types of S. alfredii leaves under Cd stress. The distinctive gene expression patterns of metal transporters indicate special gene regulatory networks underlying Cd tolerance and hyperaccumulation in S. alfredii. Collectively, our findings are the first observation of the cellular and molecular responses of S. alfredii leaves under Cd stress and lay the cornerstone for future hyperaccumulator scRNA-seq investigations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cadmium (Cd) is a prominent heavy metal pollutant that inhibits plant growth and poses risks to human health. Sedum plumbizincicola, as a Cd/Zn/Pb hyperaccumulator species, exhibits robust resistance to heavy metals and effective enrichment capacities. In our previous study, overexpressing SpbZIP60 in Arabidopsis enhanced Cd tolerance; however, the underlying the molecular mechanism remains to be elucidated. Here, we identified SpbZIP60 as a representative Cd stress response factor with nuclear localization and transcriptional activation activity. SpbZIP60 underwent conservative splicing in response to endoplasmic reticulum (ER) stress, while its response to Cd stress is independent of the ER stress-mediated unfolded protein response pathway. Overexpression of SpbZIP60 in S. alfredii increased the Cd tolerance and antioxidant activity. Furthermore, SpbZIP60 increased the content of cell wall components and thickened cell wall under Cd stress. Transcriptome analysis indicated a significant enrichment of differentially expressed genes within the phenylpropanoid metabolism pathway. Besides, the binding of SpbZIP60 to the promoter region of SpBglu resulted in the activation of gene expression, thereby enhancing the process of lignin deposition. Collectively, our results elucidated a molecular regulatory model in which SpbZIP60 increased the thickness of the root cell wall to impede Cd entry into the cell, consequently improving Cd tolerance., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

The biogeochemical processes of sulfur and heavy metals in the environment are closely related to each other. We investigated the influence of sulfur addition on hyperaccumulator Sedum alfredii Hance growth, cadmium (Cd) accumulation, soil Cd bioavailability, soil bacterial communities and plant transcriptome responses. The results showed that an appropriate rate of sulfur addition (1.0 or 2.5 g/kg) enhanced the growth of Sedum alfredii Hance plants as well as their accumulation of Cd. A high rate of sulfur addition (5.0 or 10.0 g/kg) causes toxicity to Sedum alfredii Hance plants. The application of an appropriate amount of sulfur to the soil increased the abundance of sulfur-oxidizing bacteria such as Sulfuriferula and Thiobacillus; acid-fast bacillus such as Alicyclobacillus; and cadmium-tolerant bacteria such as Bacillus and Rhodanobacter. This led to a decrease in pH and an increase in bioavailable Cd in the soil. RNA sequencing revealed that the addition of sulfur to soils led to the up regulation of most of the differentially expressed genes (DEGs) involved in "photosynthesis" and "photosynthesis, light reaction" in Sedum alfredii Hance leaves. Moreover, the "plant hormone signal transduction" pathway was significantly enriched with sulfur addition. Sulfur assimilation in Sedum alfredii Hance plants may promote photosynthesis and hormone synthesis, leading to Cd tolerance in these plants. Our study revealed that sulfur fertilization enhanced the efficiency of Cd phytoremediation in Sedum alfredii Hance plants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hyperaccumulators are the material basis and key to the phytoremediation of heavy metal contaminated soils. Conventional methods for screening hyperaccumulators are highly dependent on the time- and labor-consuming sampling and chemical analysis. In this study, a novel spectral approach assisted with multi-task deep learning was proposed to streamline accumulating ecotype screening, heavy metal stress discrimination, and heavy metals quantification in plants. The significant Cd/Zn co-hyperaccumulator Sedum alfredii and its non-accumulating ecotype were stressed by Cd, Zn, and Pb. Spectral images of leaves were rapidly acquired by hyperspectral imaging. The self-designed deep learning architecture was composed of a shallow network (ENet) for accumulating ecotype identification, and a multi-task network (HMNet) for heavy metal stress type and accumulation prediction simultaneously. To further assess the robustness of the networks, they were compared with conventional machine learning models (i.e., partial least squares (PLS) and support vector machine (SVM)) on a series of evaluation metrics of classification, multi-label classification, and regression. S. alfredii with heavy metals accumulation capability was identified by ENet with 100 % accuracy. HMNet reduced overfitting and outperformed machine learning models with the average exact match ratio (EMR) of heavy metal stress discrimination increased by 7.46 %, and residual prediction deviations (RPD) of heavy metal concentrations prediction increased by 53.59 %. The method succeeded in rapidly and accurately discriminating heavy metal stress with EMRs over 91 % and accuracies over 96 %, and in predicting heavy metals accumulation with an average RPD of 3.29 for Zn, 2.57 for Cd, and 2.53 for Pb, indicating the satisfactory practicability and potential for sensing heavy metals accumulation. This study provides a relatively novel spectral method to facilitate hyperaccumulator screening and heavy metals accumulation prediction in the phytoremediation process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a heavy metal pollutant mainly originating from the discharge of industrial sewage, irrigation with contaminated water, and the use of fertilizers. The phytoremediation of Cd polluted soil depends on the identification of the associated genes in hyperaccumulators. Here, a novel Cd tolerance gene (SpCTP3) was identified in hyperaccumulator Sedum plumbizincicola. The results of Cd 2+ binding and thermodynamic analyses, revealed the CXXC motif in SpCTP3 functions is a Cd 2+ binding site. A mutated CXXC motif decreased binding to Cd by 59.93%. The subcellular localization analysis suggested that SpCTP3 is primarily a cytoplasmic protein. Additionally, the SpCTP3-overexpressing (OE) plants were more tolerant to Cd and accumulated more Cd than wild-type Sedum alfredii (NHE-WT). The Cd concentrations in the cytoplasm of root and leaf cells were significantly higher (53.75% and 71.87%, respectively) in SpCTP3-OE plants than in NHE-WT. Furthermore, malic acid levels increased and decreased in SpCTP3-OE and SpCTP3-RNAi plants, respectively. Moreover, SpCTP3 interacted with malate dehydrogenase 1 (MDH1). Thus, SpCTP3 helps regulate the subcellular distribution of Cd and increases Cd accumulation when it is overexpressed in plants, ultimately Cd tolerance through its interaction with SpMDH1. This study provides new insights relevant to improving the Cd uptake by Sedum plumbizincicola., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

With the acceleration of industrialization, Cd pollution has emerged as a major threat to soil ecosystem health and food safety. Hyperaccumulating plants like Sedum alfredii Hance are considered to be used as part of an effective strategy for the ecological remediation of Cd polluted soils. This study delved deeply into the physiological, transcriptomic, and metabolomic responses of S. alfredii under cadmium (Cd) stress when treated with exogenous salicylic acid (SA). We found that SA notably enhanced the growth of S. alfredii and thereby increased absorption and accumulation of Cd, effectively alleviating the oxidative stress caused by Cd through upregulation of the antioxidant system. Transcriptomic and metabolomic data further unveiled the influence of SA on photosynthesis, antioxidant defensive mechanisms, and metal absorption enrichment pathways. Notably, the interactions between SA and other plant hormones, especially IAA and JA, played a central role in these processes. These findings offer us a comprehensive perspective on understanding how to enhance the growth and heavy metal absorption capabilities of hyperaccumulator plants by regulating plant hormones, providing invaluable strategies for future environmental remediation efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Silicon (Si) effectively promote the yield of many crops, mainly due to its ability to enhance plants resistance to stress. However, how Si helps hyperaccumulators to extract Cadmium (Cd) from soil has remained unclear. In this study, Sedum alfredii Hance (S. alfredii) was used as material to study how exogenous Si affected biomass, Cd accumulation, antioxidation, cell ultrastructure, subcellular distribution and changes in gene expression after Cd exposure. The study has shown that as Si concentration increases (1, 2 mM), the shoot biomass of plants increased by 33.1%-63.6%, the Cd accumulation increased by 31.9%-96.6%, and the chlorophyll, carotenoid content, photosynthetic gas exchange parameters significantly increased. Si reduced Pro and MDA, promoted the concentrations of SOD, CAT and POD to reduce antioxidant stress damage. In addition, Si promoted GSH and PC to chelate Cd in vacuoles, repaired damaged cell ultrastructure, improved the fixation of Cd and cell wall (especially in pectin), and reduced the toxic effects of Cd. Transcriptome analysis found that genes encoding Cd detoxification, Cd absorption and transport were up-regulated by Si supplying, including photosynthetic pathways (PSB, LHCB, PSA), antioxidant defense systems (CAT, APX, CSD, RBOH), cell wall biosynthesis such as pectinesterase (PME), chelation (GST, MT, NAS, GR), Cd absorption (Nramp3, Nramp5, ZNT) and Cd transport (HMA, PCR). Our result revealed the tentative mechanism of Si promotes Cd accumulation and enhances Cd tolerance in S. alfredii, and thereby provides a solid theoretical support for the practical use of Si fertilizer in phytoextraction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As an essential element, zinc (Zn) can improve or inhibit the growth of plants depending on its concentrations. In this study, the effects of 24-Epibrassinolide (EBR), one well-known steroid phytohormone regulating plant growth and alleviating abiotic stress damage, on morphological parameters and antioxidant capacities of Sedum lineare were investigated under different Zn doses. Compared to plants only exposed to Zn, simultaneously foliar application of 0.75 μM EBR significantly improved multiple morphological characteristics and such growth-improving effects were more significant at high Zn concentrations. At a detrimental 800 μM Zn, EBR benefitted plant growth most prominently, as shown by that the stem length, fresh weight and internode length were increased by 111%, 85% and 157%, respectively; than Zn solely treated plants. EBR spray also enhanced both the activities of antioxidant enzymes such as peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and the contents of antioxidative agents including ascorbic acid (AsA) and glutathione (GSH), which in turn decreased the accumulation of reactive oxygen species (ROS) and alleviated the lipid peroxidation in plants. Thus, by demonstrating that EBR could help S. lineare resist high-zinc stress through strengthening the antioxidant system, this work provided a new idea for expanding the planting range of Crassulaceae plants in heavy metal contaminated soil for phytoremediation purpose in the future., Competing Interests: The authors have declared that no competing interests exist.

Application of activating agents can significantly improve efficiency of plants for remediation of soils contaminated by heavy metals, however, damage to soil and plants limits application of traditional activating agents. The aim of our experiments is to select an efficient,green and low-cost activating agent to improve efficiency of plant extraction technology. In this study, contaminated soil was remediated by Sedum alfredii. The effects of two plant extracts (i.e., Oxalis corniculata,OX and Medicago sativaextract, ME) in addition to citric acid (CA) were studied in oscillatory activation experiment and pot experiment. The oscillation activation experiment revealed that extraction quantity of heavy metals in the soil was enhanced significantly with concentration of plant extract. The extraction quantity of Zn from 100% OX extract and ME extract were significantly higher than 10 mmol/L CA (54.04% and 33.09%, respectively). The 10 mmol/L CA has best extraction efficiency for Cd, up to 41.36 µg/kg, which is significantly higher than CK (control) (p < 0.05). The pot experiment exhibited that application of CA has significantly reduced soil pH and organic matter content by 8.63% and 28.21%, respectively, however the two extracts have no significant effect on soil properties. The study indicated that application of CA has negative effects on root morphological parameters and chlorophyll fluorescence parameters of Sedum alfredii.The addition of extracts of two plants have not caused any harm to Sedum alfredii. The application of three activating agents was beneficial for purification of Cd and Zn in soils, and its repairing efficiency was improved by 3.92, 3.37, 3.33 times and 0.44, 0.20, 0.86 times, respectively. The combination of plant extracts and hyperaccumulators can effectively remove heavy metals from contaminated soils, which provided a theoretical basis for mitigation of pollution in soils., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The F-box genes, which form one of the largest gene families in plants, are vital for plant growth, development and stress response. However, F-box gene family in Sedum alfredii remains unknown. Comprehensive studies addressing their function responding to cadmium stress is still limited. In the present study, 193 members of the F-box gene (SaFbox) family were identified, which were classified into nine subfamilies. Most of the SaFboxs had highly conserved domain and motif. Various functionally related cis-elements involved in plant growth regulation, stress and hormone responses were located in the upstream regions of SaFbox genes. RNA-sequencing and co-expression network analysis revealed that the identified SaFbox genes would be involved in Cd stress. Expression analysis of 16 hub genes confirmed their transcription level in different tissues. Four hub genes (SaFbox40, SaFbox51, SaFbox136 and SaFbox170) were heterologously expressed in a Cd-sensitive yeast cell to assess their effects on Cd tolerance. The transgenic yeast cells carrying SaFbox40, SaFbox51, SaFbox136, or SaFbox170 were more sensitive and accumulated more cadmium under Cd stress than empty vector transformed control cells. Our results performed a comprehensive analysis of Fboxs in S. alfredii and identified their potential roles in Cd stress response.

Lateral root (LR) formation promotes plant resistance, whereas high-level ethylene induced by abiotic stress will inhibit LR emergence. Considering that local auxin accumulation is a precondition for LR generation, auxin-induced genes inhibiting ethylene synthesis may thus be important for LR development. Here, we found that auxin response factor 4 ( SaARF4 ) in Sedum alfredii Hance could be induced by auxin. The overexpression of SaARF4 decreased the LR number and reduced the vessel diameters. Meanwhile, the auxin distribution mode was altered in the root tips and PIN expression was also decreased in the overexpressed lines compared with the wild-type (WT) plants. The overexpression of SaARF4 could reduce ethylene synthesis, and thus, the repression of ethylene production decreased the LR number of WT and reduced PIN expression in the roots. Furthermore, the quantitative real-time PCR, chromatin immunoprecipitation sequencing, yeast one-hybrid, and dual-luciferase assay results showed that SaARF4 could bind the promoter of 1-aminocyclopropane-1-carboxylate oxidase 4 ( SaACO4 ), associated with ethylene biosynthesis, and could downregulate its expression. Therefore, we concluded that SaARF4 induced by auxin can inhibit ethylene biosynthesis by repressing SaACO4 expression, and this process may affect auxin transport to delay LR development.

Root exudates are the most direct manifestation of the response of plants changes in the external environment. Therefore, based on non-targeted gas chromatography-time-of-flight mass spectrometry and metabolomics, the response of Sedum plumbizincicola root exudates to Cd stress was used to reveal the possible mechanism of resistance to or accumulation of Cd. The results showed that Cd significantly changed the composition and contents of S. plumbizincicola root exudates. A total of 155 metabolites were identified in S. plumbizincicola root exudates, among which 33 showed significant differences under Cd stress, including organic acids, amino acids, lipids, and polyols. Cd stress suppressed organic acid metabolism and lipid metabolism in S. plumbizincicola and significantly affected amino acid metabolism. There were 16 metabolic pathways related to Cd stress, among which arginine and proline metabolism, valine, leucine, and isoleucine biosynthesis, glycine, serine, and threonine metabolism, glutathione metabolism, and purine metabolism were the key pathways with the highest correlation, and were closely related to the stress resistance of S. plumbizincicola., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cd is one of the potential toxic elements (PTEs) exerting great threats on the environment and living organisms and arising extensive attentions worldwide. Sedum alfredii Hance, a Cd hyperaccumulator, is of great importance in studying the mechanisms of Cd hyperaccumulation and has potentials for phytoremediation. ATP-binding cassette sub-family C (ABCC) belongs to the ABC transporter family, which is deemed to closely associate with multiple physiological processes including cellular homeostasis, metal detoxification, and transport of metabolites. In the present work, ten ABCC proteins were identified in S. alfredii Hance, exhibiting uniform domain structure and divergently clustering with those from Arabidopsis. Tissue-specific expression analysis indicated that some SaABCC genes had significantly higher expression in roots (Sa23221 and Sa88F144), stems (Sa13F200 and Sa14F98) and leaves (Sa13F200). Co-expression network analysis using these five SaABCC genes as hub genes produced two clades harboring different edge genes. Transcriptional expression profiles responsive to Cd illustrated a dramatic elevation of Sa14F190 and Sa18F186 genes. Heterologous expression in a Cd-sensitive yeast cell line, we confirmed the functions of Sa14F190 gene encoding ABCC in Cd accumulation. Our study performed a comprehensive analysis of ABCCs in S. alfredii Hance, firstly mapped their tissue-specific expression patterns responsive to Cd stress, and characterized the roles of Sa14F190 genes in Cd accumulation.

To explore the effects of some chemical amendments on the plant growth and phytoextraction efficiencies of cadmium (Cd)/zinc (Zn) hyper accumulator Sedum plumbizincicola in acid soils with high aluminum (Al) toxicity, a greenhouse pot experiment was conducted. Different kinds and dosages of amendments including calciummagnesium-phosphorus fertilizer (CMP), magnesium carbonate (MgCO₃), potassium dihydrogen phosphate (KH₂POPO₄ ) were added. The results showed that CMP and MgCO₃ increased soil pH and decreased soil exchangeable Al concentration to some extent, while KH₂PO₄ reduced soil exchangeable Al concentration but had little effect on increasing soil pH. Proper application (9.39 mg/kg) of CMP could improve the biomass and Cd and Zn phytoextraction efficiencies by S. plumbizincicola but it would inhibit plant growth and phytoextraction performance when exceeding 9.39 mg/kg. MgCO₃ addition enhanced plant metal uptake while KH₂PO₄ presented an opposite effect. It suggests that using CMP and MgCO₃ could alleviate Al toxicity to S. plumbizincicola in acid soils and maintain relatively high metal extraction efficiency.

Hyperaccumulators can hyper-accumulate and -tolerate heavy metals, thus are not only an ideal model to explore the mechanisms of ion transport and toxicity tolerance, but also play an irreplaceable role in the development and application of phytoremediation. Sedum plumbizincicola is a recently identified cadmium (Cd)/zinc (Zn) hyperaccumulator in the Crassulaceae family in China. Here we report the construction and screening of its yeast-expressing cDNA library. We identified a metallothionein protein encoding gene SpMT2. SpMT2 is localized in yeast cytoplasm and expression of it in yeast specifically enhanced resistance to Cd. Further analysis showed that SpMT2 did not affect Cd absorption in yeast, but greatly inhibited Cd transport into vacuoles, indicating that SpMT2 may reduce Cd toxicity via chelation in cytoplasm. qRT-PCR analyses indicated that SpMT2 was highly expressed both in roots and shoots, and did not respond to Cd treatment. Taking together the results that SpMT2 was also cytoplasm-localized in plants, we proposed that SpMT2 may chelate/detoxify Cd and retain the complex in cytosol, which renders higher mobility of Cd thus promoting long-distance Cd transport in S. plumbizincicola.

Comparative impact of CO 2 application and endophyte inoculation was investigated on the growth, rhizosphere characteristics, and cadmium (Cd) absorption of two ecotypes of Sedum alfredii Hance in response to Cd stress under hydroponic or rhizo-box culture conditions. The results showed that both CO 2 application and endophyte inoculation significantly (P < 0.05) promoted plant growth (fresh weight and dry weight), improved root morphological properties (SRL, SRA, SRV, ARD and RTN) and exudation (pH, TOC, TN, soluble sugar and organic acids), changed Cd uptake and distribution of both ecotypes of S. alfredii. Meanwhile soil total and DTPA extractable Cd in rhizo-box decreased by biofortification treatments. Superposition biofortification exhibits utmost improvement for the above mentioned parameters, and has potential for enhancing phytoremediation efficiency of hyperaccumulator and sustaining regular growth of non-hyperaccumulator in Cd contaminated soils., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Key Message: We compared the transcriptomes of parenchymal and vascular cells of Sedum alfredii stem under Cd stress to reveal gene regulatory networks underlying Cd hyperaccumulation. Cadmium (Cd) hyperaccumulation in plants is a complex biological process controlled by gene regulatory networks. Efficient transport through vascular systems and storage by parenchymal cells are vital for Cd hyperaccumulation in the Cd hyperaccumulator Sedum alfredii, but the genes involved are poorly understood. We investigated the spatial gene expression profiles of transport and storage sites in S. alfredii stem using laser-capture microdissection coupled with RNA sequencing. Gene expression patterns in response to Cd were distinct in vascular and parenchymal cells, indicating functional divisions that corresponded to Cd transportation and storage, respectively. In vascular cells, plasma membrane-related terms enriched a large number of differentially-expressed genes (DEGs) for foundational roles in Cd transportation. Parenchymal cells contained considerable DEGs specifically concentrated on vacuole-related terms associated with Cd sequestration and detoxification. In both cell types, DEGs were classified into different metabolic pathways in a similar way, indicating the role of Cd in activating a systemic stress signalling network where ATP-binding cassette transporters and Ca 2+ signal pathways were probably involved. This study identified site-specific regulation of transcriptional responses to Cd stress in S. alfredii and analysed a collection of genes that possibly function in Cd transportation and detoxification, thus providing systemic information and direction for further investigation of Cd hyperaccumulation molecular mechanisms.

Understanding cadmium (Cd) tolerance and accumulation strategies of hyperaccumulators is crucial for promoting phytoremediation of polluted soils. Root resistance to Cd regulated by nitric oxide (NO) was investigated for the Cd hyperaccumulating ecotype (HE) of Sedum alfredii. Differed from that of its non-hyperaccumulating ecotype, Cd stress in HE roots triggered a strong NO burst mediated through both nitrate reductase and nitric oxide synthase. Elimination of endogenous NO did not affect Cd levels in roots, but greatly aggravated the metal toxicity, including increased reactive oxygen species (ROS) accumulation, oxidative damage and cell ultrastructure injury. Cadmium stress in HE triggered up-regulated SOD activities but down-regulated POD, CAT, and APX activities, which were significantly inverted by NO scavenger. The NO burst also expanded the glutathione (GSH) pool in HE roots by activation of GR, GSNOR, and γ-ECS, but had no effects on the ascorbate acid (AsA) recycle. Similar to that of NO, preferential localizations of ROS and GSH to meristem and cylinder were observed in root tips of HE. Cadmium uptake and translocation were not affected by the NO levels. These results suggest that NO burst activated a GSH-involved strategy, instead of altering Cd accumulation, to protect root tips of HE S. alfredii against Cd toxicity at early stage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A hydroponic experiment was conducted to study the interaction of Cd and Zn affecting the root morphology and accumulation of Cd and Zn in Sedum aizoon . The results show that S. aizoon exhibits a high tolerance to single stress of Cd and Zn and the concentrations of Cd and Zn in the leaf, stem, and root of S. aizoon increase as the stress levels increase. A strong phytoextraction capacity in S. aizoon was also observed for the Cd concentration in the aboveground part reaching 133.0 mg·kg -1 . The combined stress of Cd and Zn has a greater effect on the growth of S. aizoon than single stress to which the root is more sensitive. The root length, surface area, root volume, and tips significantly decrease under combined stress of Cd and Zn; the effect on the tips is most significant. The low-level Zn application (10 mg·L -1 ) has a synergistic effect on the Cd accumulation in the shoot of S. aizoon because of the promotion of Cd translocation from root to shoot. However, the addition of Cd leads to a notable antagonism of the Zn absorption of S. aizoon . The concentration ratio of Zn and Cd also has a significant effect on the Cd uptake of S. aizoon . A high ratio promotes the Cd accumulation in the shoot, while the latter is inhibited by a low Zn/Cd ratio. Therefore, S. aizoon possesses a strong potential of Cd phytoextraction and can be applied for phytoremediation of Cd-contaminated soil, while the adjustment of the ratio of Cd and Zn in the growth medium will help to enhance the phytoremediation efficiency of S. aizoon .

Plant uptake of cadmium (Cd) is affected by soil and environmental conditions. In this study, hydroponic experiments were conducted to investigate the effects of elevated CO 2 coupled with inoculated endophytic bacteria M002 on morphological properties, gas exchange, photosynthetic pigments, chlorophyll fluorescence, and Cd uptake of S. alfredii. The results showed that bio-fortification processes (elevated CO 2 and/or inoculated with endophytic bacteria) significantly (p < 0.05) promoted growth patterns, improved photosynthetic characteristics and increased Cd tolerance of both ecotypes of S. alfredii, as compared to normal conditions. Net photosynthetic rate (Pn) in intact leaves of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) were increased by 73.93 and 32.90%, respectively at the low Cd (2 μM), 84.41 and 57.65%, respectively at the high Cd level (10 μM). Superposition treatment increased Cd concentration in shoots and roots of HE, by 50.87 and 82.12%, respectively at the low Cd and 46.75 and 88.92%, respectively at the high Cd level. Besides, superposition treatment declined Cd transfer factor of NHE, by 0.85% at non-Cd rate, 17.22% at the low Cd and 22.26% at the high Cd level. These results indicate that elevated CO 2 coupled with endophytic bacterial inoculation may effectively improve phytoremediation efficiency of Cd-contaminated soils by hyperaccumulator, and alleviate Cd toxicity to non-hyperaccumulator ecotype of Sedum alfredii.

Inoculation with endophytic bacterium has been considered as a prospective application to improve the efficiency of phytoextraction. In this study, the effect of Buttiauxella sp. SaSR13 (SaSR13), a novel endophytic bacterium isolated from the root of hyperaccumulator Sedum alfredii, on plant growth and cadmium (Cd) accumulation in S. alfredii was investigated. Laser scanning confocal microscopic (LSCM) images showed that SaSR13 was mainly colonized in the root elongation and mature zones. The inoculation with SaSR13 to Cd-treated plants significantly enhanced plant growth (by 39 and 42% for shoot and root biomass, respectively), chlorophyll contents (by 38%), and Cd concentration in the shoot and root (by 32 and 22%, respectively). SaSR13 stimulated the development of roots (increased root length, surface area, and root tips number) due to an increase in the indole-3-acid (IAA) concentrations and a decrease in the concentrations of superoxide anion (O 2 .- ) in plants grown under Cd stress. Furthermore, inoculation with SaSR13 enhanced the release of root exudates, especially malic acid and oxalic acid, which might have facilitated the uptake of Cd by S. alfredii. It is suggested that inoculation with endophytic bacterium SaSR13 is a promising bioaugmentation method to enhance the Cd phytoextraction efficiency by S. alfredii.

Cadmium (Cd) is highly toxic to most organisms, but some rare plant species can hyperaccumulate Cd in aboveground tissues without suffering from toxicity. The mechanism underlying Cd detoxification by hyperaccumulators is interesting but unclear. Here, the heavy metal ATPase 3 (SpHMA3) gene responsible for Cd detoxification was isolated from the Cd/zinc (Zn) hyperaccumulator Sedum plumbizincicola. RNA interference (RNAi)-mediated silencing and overexpression of SpHMA3 were induced to investigate its physiological functions in S. plumbizincicola and a nonhyperaccumulating ecotype of Sedum alfredii. Heterologous expression of SpHMA3 in Saccharomyces cerevisiae showed Cd-specific transport activity. SpHMA3 was highly expressed in the shoots and the protein was localized to the tonoplast. The SpHMA3-RNAi lines were hypersensitive to Cd but not to Zn, with the growth of shoots and young leaves being severely inhibited by Cd. Overexpressing SpHMA3 in the nonhyperaccumulating ecotype of S. alfredii greatly increased its tolerance to and accumulation of Cd, but not Zn. These results indicate that elevated expression of the tonoplast-localized SpHMA3 in the shoots plays an essential role in Cd detoxification, which contributes to the maintenance of the normal growth of young leaves of S. plumbizincicola in Cd-contaminated soils., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Phytoremediation is an effective method to remediate Pb-contaminated soils and root exudates play an important role in this process. Based on gas chromatography-mass spectrometry (GC-MS) and metabolomics method, this study focuses on the comparative metabolic profiling analysis of root exudates from the Pb-accumulating and non-accumulating ecotypes of Sedum alfredii treated with 0 and 50 μmol/L Pb. The results obtained show that plant type and Pb stress can significantly change the concentrations and species of root exudates, and fifteen compounds were identified and assumed to be potential biomarkers. Leaching experiments showed that l-alanine, l-proline and oxalic acid have a good effect to activate Pb in soil, glyceric acid and 2-hydroxyacetic acid have a general effect to activate Pb in soil. 4-Methylphenol and 2-methoxyphenol might be able to activate Pb in soil, glycerol and diethyleneglycol might be able to stabilize Pb in soil, but these activation effect and stabilization effect were all not obvious.

The hyperaccumulating ecotype of Sedum alfredii Hance is a cadmium (Cd)/zinc/lead co-hyperaccumulating species of Crassulaceae. It is a promising phytoremediation candidate accumulating substantial heavy metal ions without obvious signs of poisoning. However, few studies have focused on the regulatory roles of miRNAs and their targets in the hyperaccumulating ecotype of S. alfredii. Here, we combined analyses of the transcriptomics, sRNAs and the degradome to generate a comprehensive resource focused on identifying key regulatory miRNA-target circuits under Cd stress. A total of 87 721 unigenes and 356 miRNAs were identified by deep sequencing, and 79 miRNAs were differentially expressed under Cd stress. Furthermore, 754 target genes of 194 miRNAs were validated by degradome sequencing. A gene ontology (GO) enrichment analysis of differential miRNA targets revealed that auxin, redox-related secondary metabolism and metal transport pathways responded to Cd stress. An integrated analysis uncovered 39 pairs of miRNA targets that displayed negatively correlated expression profiles. Ten miRNA-target pairs also exhibited negative correlations according to a real-time quantitative PCR analysis. Moreover, a coexpression regulatory network was constructed based on profiles of differentially expressed genes. Two hub genes, ARF4 (auxin response factor 4) and AAP3 (amino acid permease 3), which might play central roles in the regulation of Cd-responsive genes, were uncovered. These results suggest that comprehensive analyses of the transcriptomics, sRNAs and the degradome provided a useful platform for investigating Cd hyperaccumulation in S. alfredii, and may provide new insights into the genetic engineering of phytoremediation., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Main Conclusion: The enhanced expression of a P 1B -type ATPase gene ( SaHMA3 ) is essential for Cd hyperaccumulation and hypertolerance in Sedum alfredii Hance. A functional understanding of the mechanism through which hyperaccumulator plants accumulate and tolerate extremely toxic metals is a prerequisite for the development of novel strategies for improving phytoremediation using engineered plants or natural hyperaccumulators as well as biofortification and food crop safety. Most hyperaccumulator species, however, are small and slow-growing, and their potential for large-scale decontamination of polluted soils is limited. Sedum alfredii Hance, the only one metal hyperaccumulator from the Crassulaceae family, is an ideal candidate for gaining a functional understanding of the intra-family hyperaccumulation mechanisms as well as their potential applications. In the present study, we isolated and functionally characterized a P1B-type ATPase gene (SaHMA3) from S. alfredii Hance. SaHMA3 alleles from a hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) were constitutively expressed in both shoot and root and encoded tonoplast-localized proteins, but showed differences in transport substrate specificity and expression level. SaHMA3 h from the HE plant was a Cd transporter. In contrast, SaHMA3n from NHE plants was able to transport both Zn and Cd. SaHMA3 showed a significantly higher constitutive expression level in HE plants than in NHE plants. Furthermore, the expression level of SaHMA3 in the shoots of HE plants was considerably higher than in the roots. Overexpression of SaHMA3h in tobacco plants significantly enhanced Cd tolerance and accumulation and greatly increased the root sequestration of Cd. In summary, our data suggested that SaHMA3 plays critical roles in Cd hyperaccumulation and hypertolerance in Cd hyperaccumulator S. alfredii Hance.

The combined effects of elevated CO2 and cadmium (Cd) on photosynthetic rate, chlorophyll fluorescence and Cd accumulation in hyperaccumulator Sedum alfredii Hance were investigated to predict plant growth under Cd stress with rising atmospheric CO2 concentration. Both pot and hydroponic experiments were conducted and the plants were grown under ambient (350 µL L(-1)) or elevated (800 µL L(-1)) CO2 . Elevated CO2 significantly (P < 0.05) increased Pn (105%-149%), Pnmax (38.8%-63.0%) and AQY (20.0%-34.8%) of S. alfredii in all the Cd treatments, but reduced chlorophyll concentration, dark respiration and photorespiration. After 10 days growth in medium with 50 µM Cd under elevated CO2 , PSII activities were significantly enhanced (P < 0.05) with Pm, Fv/Fm, Φ(II) and qP increased by 66.1%, 7.5%, 19.5% and 16.4%, respectively, as compared with ambient-grown plants. Total Cd uptake in shoot of S. alfredii grown under elevated CO2 was increased by 44.1%-48.5%, which was positively correlated with the increase in Pn. These results indicate that elevated CO2 promoted the growth of S. alfredii due to increased photosynthetic carbon uptake rate and photosynthetic light-use efficiency, and showed great potential to improve the phytoextraction of Cd by S. alfredii., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Endophyte-assisted phytoremediation has recently been suggested as a successful approach for ecological restoration of metal contaminated soils, however little information is available on the influence of endophytic bacteria on the phytoextraction capacity of metal hyperaccumulating plants in multi-metal polluted soils. The aims of our study were to isolate and characterize metal-resistant and 1-aminocyclopropane-1-carboxylate (ACC) utilizing endophytic bacteria from tissues of the newly discovered Zn/Cd hyperaccumulator Sedum plumbizincicola and to examine if these endophytic bacterial strains could improve the efficiency of phytoextraction of multi-metal contaminated soils. Among a collection of 42 metal resistant bacterial strains isolated from the tissues of S. plumbizincicola grown on Pb/Zn mine tailings, five plant growth promoting endophytic bacterial strains (PGPE) were selected due to their ability to promote plant growth and to utilize ACC as the sole nitrogen source. The five isolates were identified as Bacillus pumilus E2S2, Bacillus sp. E1S2, Bacillus sp. E4S1, Achromobacter sp. E4L5 and Stenotrophomonas sp. E1L and subsequent testing revealed that they all exhibited traits associated with plant growth promotion, such as production of indole-3-acetic acid and siderophores and solubilization of phosphorus. These five strains showed high resistance to heavy metals (Cd, Zn and Pb) and various antibiotics. Further, inoculation of these ACC utilizing strains significantly increased the concentrations of water extractable Cd and Zn in soil. Moreover, a pot experiment was conducted to elucidate the effects of inoculating metal-resistant ACC utilizing strains on the growth of S. plumbizincicola and its uptake of Cd, Zn and Pb in multi-metal contaminated soils. Out of the five strains, B. pumilus E2S2 significantly increased root (146%) and shoot (17%) length, fresh (37%) and dry biomass (32%) of S. plumbizincicola as well as plant Cd uptake (43%), whereas Bacillus sp. E1S2 significantly enhanced the accumulation of Zn (18%) in plants compared with non-inoculated controls. The inoculated strains also showed high levels of colonization in rhizosphere and plant tissues. Results demonstrate the potential to improve phytoextraction of soils contaminated with multiple heavy metals by inoculating metal hyperaccumulating plants with their own selected functional endophytic bacterial strains., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hydroponic experiments were conducted to investigate the variation of root exudates from the hyperaccumulator Sedum alfredii under the stress of cadmium (Cd). S. alfredii was cultured for 4 days in the nutrient solution spiked with CdCl2 at concentrations of 0, 5, 10, 40, and 400 µM Cd after the pre-culture. The root exudates were collected and analyzed by GC-MS, and 62 compounds were identified. Of these compounds, the orthogonal partial least-squares discrimination analysis (OPLS-DA) showed that there were a distinct difference among the root exudates with different Cd treatments and 20 compounds resulting in this difference were found out. Changing tendencies in the relative content of these 20 compounds under the different Cd treatments were analyzed. These results indicated that trehalose, erythritol, naphthalene, d-pinitol and n-octacosane might be closely related to the Cd stabilization, phosphoric acid, tetradecanoic acid, oxalic acid, threonic acid and glycine could be attributed to the Cd mobilization, and mannitol, oleic acid, 3-hydroxybutanoic acid, fructose, octacosanol and ribitol could copy well with the Cd stress.

Sedum alfredii Hance is a promising cadmium (Cd) hyperaccumulating plant recently identified in China. However, the physiological and molecular mechanisms underlying Cd accumulation, which differentiate hyperaccumulating ecotype (HE) from non-hyperaccumulating ecotype (NHE) has not been elucidated yet. A hydroponic experiment was conducted to investigate the role of sulfur assimilation pathway in Cd hyperaccumulation by the S. alfredii Hance, by analyzing gene expression pattern in sulfur assimilation pathway and the concentration of some sulfur containing compounds. The results show that, sulfur assimilation pathway was affected by Cd differently in HE and NHE S. alfredii Hance. The gene expression pattern of sulfur assimilation pathway was regulated differently in HE and NHE plants, especially the nicotianamine synthase (NAS). NAS transcript levels in root of HE was 141-fold higher than NHE, while in shoots of HE only 0.31-fold higher than NHE. In HE roots, NAS expression level was maximum 3171-fold higher than shoots, while in NHE plants roots NAS expression level was maximum 45.3-fold higher than shoots. In HE plant roots, sulfur, cysteine and methionine concentrations increased 30%, 46% and 835% respectively, by Cd treatment, but in NHE plants roots, sulfur concentration increased less than 1%, cysteine and methionine concentrations decreased 78.5% and 13.3% respectively, by Cd. Cd exposure increased glutathione levels by 142% in HE but less than 10% in NHE plant roots., (© 2013 Published by Elsevier Inc.)

In order to select appropriate amendments for cropping hyperaccumulator or normal plants on contaminated soils and establish the relationship between Cd sorption characteristics of soil amendments and their capacity to reduce Cd uptake by plants, batch sorption experiments with 11 different clay minerals and organic materials and a pot experiment with the same amendments were carried out. The pot experiment was conducted with Sedum alfredii and maize (Zea mays) in a co-cropping system. The results showed that the highest sorption amount was by montmorillonite at 40.82 mg/g, while mica was the lowest at only 1.83 mg/g. There was a significant negative correlation between the n value of Freundlich equation and Cd uptake by plants, and between the logarithm of the stability constant K of the Langmuir equation and plant uptake. Humic acids (HAs) and mushroom manure increased Cd uptake by S. alfredii, but not maize, thus they are suitable as soil amendments for the co-cropping S. alfredii and maize. The stability constant K in these cases was 0.14-0.16 L/mg and n values were 1.51-2.19. The alkaline zeolite and mica had the best fixation abilities and significantly decreased Cd uptake by the both plants, with K > or = 1.49 L/mg and n > or = 3.59.

Real-time Quantitative PCR (RT-qPCR) has become an effective method for accurate analysis of gene expression in several biological systems as well as under different experimental conditions. Although with high sensitivity, specificity and broad dynamic range, this method requires suitable reference genes for transcript normalization in order to guarantee reproducible and meaningful results. In the present study, we evaluated five traditional housekeeping genes and five novel reference genes in Hyperaccumulating ecotype of Sedum alfredii, a well known hyperaccumulator for heavy metals phytoremediation, under Cd, Pb, Zn and Cu stresses of seven different durations. The expression stability of these ten candidates were determined with three programs--geNorm, NormFinder and BestKeeper. The results showed that all the selected reference genes except for SAND could be used for RT-qPCR normalization. Among them UBC9 and TUB were ranked as the most stable candidates across all samples by three programs together. For the least stable reference genes, however, BestKeeper produced different results compared with geNorm and NormFinder. Meanwhile, the expression profiles of PCS under Cd, Pb, Zn and Cu stresses were assessed using UBC9 and TUB respectively, and similar trends were obtained from the results of the two groups. The distinct expression patterns of PCS indicated that various strategies could be taken by plants in adaption to different heavy metals stresses. This study will provide appropriate reference genes for further gene expression quantification using RT-qPCR in Hyperaccumulator S. alfredii.

Non-accumulated metals in mixed metal contaminated soils may affect hyperaccumulator growth and metal accumulation and thus remediation efficiency. Two hydroponics experiments were conducted to investigate the effects of copper (Cu) on cadmium (Cd) and zinc (Zn) accumulation by the Cd/Zn hyperaccumulator Sedum plumbizincicola, Cu toxicity and plant detoxification using chemical sequential extraction of metals, sub-cellular separation, micro synchrotron radiation based X-ray fluorescence, and transmission electron microscopy. Compared with the control (0.31 μM Cu), 5-50 μM Cu had no significant effect on Cd/Zn accumulation, but Cu at 200 μM induced root cell plasmolysis and disordered chloroplast structure. The plants held Cu in the roots and cell walls and complexed Cu in insoluble forms as their main detoxification mechanisms. Exposure to 200 μM Cu for 4 days inhibited plant Cd uptake and translocation but did not affect Zn concentrations in roots and stems. Moreover, unloading of Cd and Zn from stem to leaf was restrained compared to control plants, perhaps due to Cu accumulation in leaf veins. Copper may thus interfere with root Cd uptake and restrain Cd/Zn unloading to the leaves. Further investigation of how Cu affects plant metal uptake may help elucidate the Cd/Zn hyper-accumulating mechanisms of S. plumbizincicola., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The Sedum alfredii Hance hyperaccumulating ecotype (HE) has the ability to hyperaccumulate cadmium (Cd), as well as zinc (Zn) and lead (Pb) in above-ground tissues. Although many physiological studies have been conducted with these plants, the molecular mechanisms underlying their hyper-tolerance to heavy metals are largely unknown. Here we report on the generation of 9.4 gigabases of adaptor-trimmed raw sequences and the assembly of 57,162 transcript contigs in S. alfredii Hance (HE) shoots by the combination of Roche 454 and Illumina/Solexa deep sequencing technologies. We also have functionally annotated the transcriptome and analyzed the transcriptome changes upon Cd hyperaccumulation in S. alfredii Hance (HE) shoots. There are 110 contigs and 123 contigs that were up-regulated (Fold Change ≥ 2.0) and down-regulated (Fold Change

The effects of elevated CO2 on the metal bioavailability and the rhizosphere characteristics of hyperaccumulator are not well understood. In this study, soil pot experiment was carried out to contrast the effects of elevated CO2 on rhizosphere characteristics between a hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of Sedum alfredii grown under ambient (350 μL L(-1)) or elevated (800 μL L(-1)) CO2. Elevated CO2 facilitated the growth of both ecotypes of S. alfredii, but the promotion in the HE was much greater than in the NHE. No significant (P<0.05) changes in soil pH, dissolved organic matter (DOM) and microbial biomass (Cmic) were observed in the rhizosphere of NHE under both CO2 level. For HE, however, elevated CO2 reduced soil pH by 0.3 units, increased DOM (especially for hydrophilic acid (HiA) fractions) by 19.2% and Cmic by 19%, as compared to ambient CO2. Mobile Cd and Zn (extractable with 1M NH4NO3) in the rhizosphere of HE decreased considerably, but the decreases were greater under ambient CO2 than under elevated CO2. Phytoextraction efficiency of Cd and Zn by HE was increased significantly by elevated CO2 (P<0.05). The results suggest that elevated CO2 can change soil microenvironment, increase bioavailability of Cd and Zn and thus facilitate metal uptake by the HE. This work highlights that elevated CO2 may be a useful way to improve phytoremediation efficiency of Cd/Zn-contaminated soil by hyperaccumulating ecotype S. alfredii., (Copyright © 2013 Elsevier B.V. All rights reserved.)

While phytoextraction tools are increasingly applied to remediation of contaminated soils, strategies are needed to optimize plant uptake by improving soil conditions. Mineral nutrition affects plant growth and metal absorption and subsequently the accumulation of heavy metal through hyper-accumulator plants. Microcosm experiments were conducted in greenhouse to examine the effect of different phosphorus (P) sources on zinc (Zn) phytoextraction by Sedum alfredii in aged Zn-contaminated paddy soil. The Zn accumulation, soil pH, microbial biomass and enzyme activity, available Zn changes. and Zn phytoremediation efficiency in soil after plant harvest were determined. Upon addition of P, Zn uptake of S. alfredii significantly increased. Mehlich-3 extractable or the fractions of exchangeable and carbonate-bound soil Zn were significantly increased at higher P applications. Soil pH significantly decreased with increasing P application rates. Soil microbial biomass in the P-treated soils was significantly higher (P < 0.05) than those in the control. Shoot Zn concentration was positively correlated with Mehlich-3 extractable P (P < 0.0001) or exchangeable/carbonate-bound Zn (P < 0.001), but negatively related to soil pH (P < 0.0001). These results indicate that application of P fertilizers has the potential to enhance Zn mobility and uptake by hyperaccumulating plant S. alfredii, thus increasing phytoremediation efficiency of Zn-contaminated soils.

In contrast to the well-documented roles of its mono- and bisphosphate esters, the occurrence of free sedoheptulose in plant tissues remains a matter of conjecture. The present work sought to determine the origin of sedoheptulose formation in planta, as well as its physiological importance. Elevated CO2 and sucrose induction experiments were used to study sedoheptulose metabolism in the Crassulacean acid metabolism (CAM) plants Kalanchoë pinnata and Sedum spectabile. Experimental evidence suggested that sedoheptulose is produced from the oxidative pentose phosphate pathway intermediate sedoheptulose-7-phosphate, by a sedoheptulose-7-phosphate phosphatase. Carbon flux through this pathway was stimulated by increased triose-phosphate levels (elevated CO2, compromised sink availability, and sucrose incubation of source leaves) and attenuated by ADP and inorganic phosphate (Pi). The accumulation of free sedoheptulose is proposed to act as a mechanism contributing to both C and P homeostasis by serving as an alternative carbon store under elevated CO2 or a compromised sink capacity to avoid sucrose accumulation, depletion of inorganic phosphate, and suppression of photosynthesis. It remains to be established whether this acclimation-avoiding mechanism is confined to CAM plants, which might be especially vulnerable to Pi imbalances, or whether some C3 and C4 plants also dispose of the genetic capacity to induce and accelerate sedoheptulose synthesis upon CO2 elevation.

The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils. Organic acid has been suggested to be involved in toxic metallic element tolerance, translocation, and accumulation in plants. The impact of exogenous organic acids on cadmium (Cd) uptake and translocation in the zinc (Zn)/Cd co-hyperaccumulator Sedum alfredii was investigated in the present study. By the addition of organic acids, short-term (2 h) root uptake of (109)Cd increased significantly, and higher (109)Cd contents in roots and shoots were noted 24 h after uptake, when compared to controls. About 85% of the (109)Cd taken up was distributed to the shoots in plants with citric acid (CA) treatments, as compared with 75% within controls. No such effect was observed for tartaric acid (TA). Reduced growth under Cd stress was significantly alleviated by low CA. Long-term application of the two organic acids both resulted in elevated Cd in plants, but the effects varied with exposure time and levels. The results imply that CA may be involved in the processes of Cd uptake, translocation and tolerance in S. alfredii, whereas the impact of TA is mainly on the root uptake of Cd.

Sedum alfredii Hance is a fast-growing and high-biomass zinc (Zn) hyperaccumulator native to China. A compound containing substituted indole ligand was isolated from this Zn hyperaccumulator plants by sonication/ethanol extraction, macroporous resin column as well as preparative HPLC (P-HPLC). Hydroponic experiment showed that the concentrations of both Zn and the compound containing substituted indole ligand were remarkably increased in stems and leaves of both hyperaccumulator and non-hyperaccumulator as Zn rising from 0.5 to 50 micromol L(-1), with much more in the stems of hyperaccumulator than non-hyperaccumulator. At 50 micromol L(-1) Zn, hyperaccumulator grew normally but its non-hyperaccumulator suffered from strongly Zn-induced toxicity. This suggested that there was a positive correlation between the compound containing substituted indole ligand and Zn concentration in shoots of hyperaccumulator S. alfredii.

Organic materials with different functional groups can be used to enhance metal bioavailability. Traditional organic materials (rice straw and clover) and ethylenediamine disuccinic acid (EDDS) were applied to enhance metal uptake from polluted soil by Sedum plumbizincicola after repeated phytoextraction. Changes in pH, dissolved organic carbon (DOC) and metal concentrations were determined in the soil solution after EDDS application. Amendment of the soil with ground rice straw or ground clove resulted in higher concentrations of Cd only (by factors of 1.92 and 1.71 respectively) in S. plumbizincicola compared to control soil. Treatment with 3 mmol kg(-1) EDDS increased all the metals studied by factors of 60.4, 1.67, and 0.27 for Cu, Cd, and Zn, respectively. EDDS significantly increased soil solution DOC and pH and increased soil plant-available metals above the amounts that the plants could take up, resulting in high soil concentrations of soluble metals and high risk of ground water contamination. After repeated phytoremediation of metal contaminated soils the efficiency of metal removal declines as the concentrations of bioavailable metal fractions decline. Traditional organic materials can therefore be much more effective and environmentally friendly amendments than EDDS in enhancing phytoremediation efficiency of Cd contaminated soil

Background, Aim and Scope: Lead (Pb) accumulation in soils affects plants primarily through their root systems. The aim of this study was to investigate early symptoms of the loss of membrane integrity and lipid peroxidation in root tissues and physiological adaptation mechanism to Pb in accumulating ecotypes (AE) and non-accumulating ecotypes (NAE) of Sedum alfredii under Pb stress in hydroponics., Methods and Results: Histochemical in situ analyses, fluorescence imaging, and normal physiological analysis were used in this study. Pb accumulation in roots of both AE and NAE increased linearly with increasing Pb levels (0-200 μM), and a significant difference between both ecotypes was noted. Both loss of plasma membrane integrity and lipid peroxidation in root tissues became serious with increasing Pb levels, maximum tolerable Pb level was 25 and 100 μM for NAE and AE, respectively. Pb supplied at a toxic level caused a burst of reactive oxygen species (ROS) in root cells in both ecotypes. However, the root cells of AE had inherently higher activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), and lipoxygenase (LOX) in control plants, and the induction response of these antioxidant enzymes occurred at lower Pb level in AE than NAE. AE plants maintained higher ascorbic acid and H(2)O(2) concentrations in root cells than NAE when exposed to different Pb levels, and Pb induced more increase in dehydroascorbate (DHA), catalase (CAT), and ascorbate peroxidase (APX) in AE than NAE roots., Discussion and Conclusion: Results indicate that histochemical in situ analyses of root cell death and lipid peroxidation under Pb short-term stress was sensitive, reliable, and fast. Higher tolerance in roots of accumulating ecotype under Pb stress did depend on effective free oxygen scavenging by making complex function of both constitutively higher activities and sensitive induction of key antioxidant enzymes in root cells of S. alfredii.

Improving the efficacy of phytoextraction is critical for its successful application in metal contaminated soils. Mineral nutrition affects plant growth and metal absorption and subsequently the accumulation of heavy metal through hyper-accumulator plants. This study assessed the effects of di-hydrogen phosphates (KH2PO4, Ca(H2PO4)2, NaH2PO4 and NH4H2PO4) application at three levels (22, 88 and 352 mg P/kg soil) on Sedum alfredii growth and metal uptake by three consecutive harvests on aged and Zn/Cd combined contaminated paddy soil. The addition of phosphates (P) significantly increased the amount of Zn taken up by S. alfredii due to increased shoot Zn concentration and dry matter yield (DMY) (P < 0.05). The highest phytoextraction of Zn and Cd was observed in KH2PO4 and NH4H2PO4 treatment at 352 mg P/kg soil. The amount of Zn removed by phytoextraction increased in the order of 1st clipping < 2nd clipping < 3rd clipping, and for Cd extraction the order was 2nd clipping < 1st clipping < 3rd clipping. These results indicate that the application of P fertilizers coupled with multiple cuttings can enhance the removal of Zn and Cd from contaminated soils by S. alfredii, thus shortening the time needed for accomplishing remediation goals.

Spatial imaging of cadmium (Cd) in the hyperaccumulator Sedum alfredii was investigated in vivo by laser ablation inductively coupled plasma mass spectrometry and x-ray microfluorescence imaging. Preferential Cd accumulation in the pith and cortex was observed in stems of the Cd hyperaccumulating ecotype (HE), whereas Cd was restricted to the vascular bundles in its contrasting nonhyperaccumulating ecotype. Cd concentrations of up to 15,000 μg g(-1) were measured in the pith cells, which was many fold higher than the concentrations in the stem epidermis and vascular bundles in the HE plants. In the leaves of the HE, Cd was mainly localized to the mesophyll and vascular cells rather than the epidermis. The distribution pattern of Cd in both stems and leaves of the HE was very similar to calcium but not zinc, irrespective of Cd exposure levels. Extended x-ray absorption fine structure spectroscopy analysis showed that Cd in the stems and leaves of the HE was mainly associated with oxygen ligands, and a larger proportion (about 70% in leaves and 47% in stems) of Cd was bound with malic acid, which was the major organic acid in the shoots of the plants. These results indicate that a majority of Cd in HE accumulates in the parenchyma cells, especially in stems, and is likely associated with calcium pathways and bound with organic acid (malate), which is indicative of a critical role of vacuolar sequestration of Cd in the HE S. alfredii.

Pot experiments were conducted to investigate the changes of the dissolved organic matter (DOM) in the rhizosphere of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of Sedum alfredii and its effects on Zn and Cd sorption by soils. After planted with HE, soil pH in the rhizosphere reduced by 0.5-0.6 units which is consistent with the increase of DOM. The hydrophilic fractions (51%) in DOM from the rhizosphere of HE (HE-DOM) was much greater than NHE-DOM (35%). In the presence of HE-DOM, Zn and Cd sorption capacity decreased markedly in the following order: calcareous clay loam>neutral clay loam>acidic silty clay. The sorption isotherms could be well described by the Freundlich equation (R(2)>0.95), and the partition coefficient (K) in the presence of HE-DOM was decreased by 30.7-68.8% for Zn and 20.3-59.2% for Cd, as compared to NHE-DOM. An increase in HE-DOM concentration significantly reduced the sorption and increased the desorption of Zn and Cd by three soils. DOM derived from the rhizosphere of the hyperaccumulating ecotype of S. alfredii could significantly reduce metal sorption and increase its mobility through the formation of soluble DOM-metal complexes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The Zn/Cd hyperaccumulating ecotype (HE) of Sedum alfredii Hance can accumulate 24- and 28-fold higher leaf and stem Zn concentrations when compared with the non-hyperaccumulating ecotype (NHE) of Sedum. Heterologous expression of a metal tolerance protein (MTP1) encoding gene from HE plants (SaMTP1) or the homologous gene from NHE plants (SnMTP1) suppressed Zn(2+) hypersensitivity in the Δzrc1 yeast mutant. In plants, SaMTP1 localized to the tonoplast. Furthermore, MTP1 transcript level in the shoot of HE plants was more than 80-fold higher than that of NHE plants. The transcript level of SaMTP1 in shoot was up-regulated 1-fold by Zn(2+) while the expression of SnMTP1 was slightly inhibited. These data suggest that SaMTP1 can play an important role in Zn accumulation in HE plants., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Phytoextraction using hyperaccumulating plants is generally time-consuming and requires the cessation of agriculture. We coupled chelators and a co-cropping system to enhance phytoextraction rates, while allowing for agricultural production. An experiment on I m3 lysimeter beds was conducted with a co-cropping system consisting of the hyperaccumulator Sedum alfredii and low-accumulating corn (Zea Mays, cv. Huidan-4), with addition ofa mixture of chelators (MC), to assess the efficiency of chelator enhanced co-crop phytoextraction and the leaching risk caused by the chelator. The results showed that the addition of MC promoted the growth of S. alfredii in the first crop (spring-summer season) and significantly increased the metal phytoextraction. The DTPA-extractable and total metal concentrations in the topsoil were also reduced more significantly with the addition of MC compared with the control treatments. However, mono-cropped S. alfredii without MC was more suitable for maximizing S. alfredii growth and therefore phytoextraction of Zn and Cd during the autumn-winter seasons. No adverse impact to groundwater due to MC application was observed during the experiments with three crops and three MC applications. But elevated total Cd and Pb concentrations among subsoils compared to the initial subsoil concentrations were found for the co-crop + MC treatment after the third crop.

Sedum alfredii is a well-known Cd (cadmium) hyperaccumulator native to China. The impacts of exogenous Ca on Cd-induced oxidative stress and antioxidant systems in roots of S. alfredii were investigated by using cellular and biochemical approaches. Supplementation of the medium with higher Ca levels resulted in alleviated growth inhibition and decreased Cd concentration, as well as increased Ca concentration in roots. Cadmium induced lipid peroxidation and loss of plasma membrane integrity, reactive oxygen species overproduction, as well as ultrastructural changes of root cells were largely reversed by Ca supplementation in the medium. Calcium application significantly altered the Cd effects on antioxidant enzymes and non-enzyme antioxidants (non-protein thiols), and significantly increased glutathione (GSH) biosynthesis. The results suggest that Ca is able to protect the roots of S. alfredii against Cd toxicity by restoration of Cd-displaced Ca, alleviation of the metal induced oxidative stress, as well as promotion of GSH biosynthesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance.

* Sedum alfredii is a fast-growing, high-biomass zinc (Zn) hyperaccumulator native to China. Here, the characteristics of in vivo Zn distribution in stems and leaves of the hyperaccumulating (HE) and nonhyperaccumulating ecotypes (NHE) of S. alfredii were investigated by synchrotron radiation X-ray fluorescence (SRXRF) analysis, together with a Zn probe. * Preferential Zn accumulation in leaf and stem epidermis was observed in both ecotypes, but to a much greater extent for HE. Epidermal Zn increased largely in leaves and stems of HE as exposure time was prolonged, while Zn saturation occurred relatively early in HE leaf mesophyll cells and stem vascular bundles. A second peak of Zn enrichment in stem and leaf vascular systems was shown in both ecotypes. However, the proportion of Zn accumulated in stem vascular bundles relative to other tissues was much greater for HE than for NHE. * Leaf and stem distribution patterns of phosphorus (P) and sulphur (S) in the HE were very like that for Zn, while the calcium (Ca) distribution pattern was the reverse of that for Zn. No such relationship was observed in NHE. * Our study mainly suggested that epidermal layers serve as important storage sites for accumulated Zn in the S. alfredii HE.

In this study, the heavy metal contents were detected in plants of Sedum alfredii and soils from Pb/Zn mined area and non-mined area, and a dendrogram was generated by using RAPD methods based on the hyperaccumulating ecotype (HE), the non-hyperaccumulating ecotype (NHE) and other species of Sedum. The results showed that the available Pb of the Pb/Zn mined soil was 77-fold higher, and available Zn and Cd were 10-fold and 16-fold higher in the mined soil than in the non-mined soil, respectively. The dendrogram showed that the HE S. alfredii was the nearest relative to NHE S. alfredii. However, genomic variation of two ecotypes was still notable, indicating that heavy metal stress had great impacts on the genetic diversity and plant evolution, and HE may be a mutant from the NHE. Ten RAPD bands were observed only in the HE as compared with other species of Sedum. The character of Zn/Cd hyperaccumulation in HE appeared to be related to SH-containing compounds and resist osmotic stress, and also many unknown genes.

Zn phytotoxicity and its possible detoxifying responses in two ecotypes of Sedum alfredii Hance, i.e. hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) were investigated. HE grew better with high Zn concentrations of 29.11gkg(-1) DW in shoots when exposed to 500microM Zn2+. Toxicity symptoms caused by Zn in root cells of both ecotypes mainly included plasmolysis, disruption of plasma membranes and increased cell vacuolation. At high supplied Zn concentration, chloroplasts suffered from structural disorganization in both ecotypes. Zn-induced hydrogen peroxide (H2O2) and superoxide radical (O(2)-) productions in leaves were determined by a histochemical method, which revealed that Zn stress may have involved NADPH oxidase, protein phosphatases and intracellular Ca2+ to activate the reactive oxygen species production. Inhibition of glutathione synthesis may have led to increased H2O2 and O(2)- accumulations in leaves of HE. In response to higher Zn concentrations, ascorbic acid significantly increased in both ecotypes and levels of glutathione increased in both leaves and roots of HE and in roots of NHE without any change in the leaves of NHE. The enzymatic activities like those of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2) in leaves of HE were all enhanced at supplied Zn concentration of 500microM, which may account for its better growth.