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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.)

Intercropping with hyperaccumulators can facilitate the safe utilization of cadmium-contaminated soil. However, the effectiveness of this approach is influenced by plant species and varieties, which necessitates research on optimal plant consortia. In this study, 8 tomato varieties (3 cherry tomatoes and 5 common large-fruit tomatoes) were intercropped with Sedum alfredii in a moderately Cd-contaminated vegetable field. The results showed that the Cd concentration in the fruits of common large-fruit tomato varieties under monoculture was 1.03-1.50 mg/kg, while that in the fruits of cherry tomato varieties was 0.67-0.71 mg/kg. After intercropping with S. alfredii, the fruit Cd concentrations of Hangza 501, Hangza 503, and Hangza 108 decreased by 16.42 %, 19.72 %, and 6.76 %, respectively, while those of the other varieties significantly increased, except for those of Hangza 8. In contrast, the shoot Cd concentration of cherry tomatoes was greater than that of large-fruit tomatoes under monoculture. Furthermore, a significant increase in the shoot Cd concentration was noted in the Hangza 501, Hangza 503 and Hangza 603 plants following intercropping. Additionally, intercropping with S. alfredii increased the concentration of soluble sugars in the fruits of Hangza 8, Hangza 501, Hangza 503 and Hangza 603 by 4.66 %, 17.91 %, 10.60 % and 17.88 %, respectively. Intercropping with tomatoes resulted in a decrease in both the biomass and Cd uptake of S. alfredii. Interestingly, the inhibitory effect on S. alfredii was less pronounced when intercropped with cherry tomatoes than when intercropped with large-fruit tomatoes. Among the intercropping treatments, S. alfredii exhibited the greatest total Cd accumulation (0.06 mg/plant) when intercropped with Hangza 503. In conclusion, the cherry tomato variety Hangza 503 was the most suitable for intercropping with S. alfredii and can be used safely for vegetable production and simultaneous phytoremediation of polluted soil. Our findings suggest that strategic selection of tomato varieties can optimize the effectiveness of "phytoextraction coupled with agro-safe production" technology for managing soil Cd concentrations., 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.)

Endophytic fungus Serendipita indica can bolster plant growth and confer protection against various biotic and abiotic stresses. However, S. indica - reshaped rhizosphere microecology interactions and root-soil interface processes in situ at the submicrometer scale remain poorly understood. We combined amplicon sequencing and high-resolution nano X-ray fluorescence (nano-XRF) imaging of the root-soil interface to reveal cadmium (Cd) rhizosphere processes. S. indica can successfully colonize the roots of Sedum alfredii Hance, which induces a remarkable increase in shoot biomass by 211.32% and Cd accumulation by 235.72%. Nano-XRF images showed that S. indica colonization altered the Cd distribution in the rhizosphere and facilitated the proximity of more Cd and sulfur (S) to enter the roots and transport to the shoot. Furthermore, the rhizosphere-enriched microbiota demonstrated a more stable network structure after the S. indica inoculation. Keystone species were strongly associated with growth promotion and Cd absorption. For example, Comamonadaceae are closely related to the organic acid cycle and S bioavailability, which could facilitate Cd and S accumulation in plants. Meanwhile, Sphingomonadaceae could release auxin and boost plant biomass. In summary, we construct a mutualism system for beneficial fungi and hyperaccumulation plants, which facilitates high-efficient remediation of Cd-contaminated soils by restructuring the rhizosphere microbiota.

To increase the efficiency of phytoremediation to clean up heavy metals in soil, assisted with alternating current (AC) electric field technology is a promising choice. Our experiments utilized the hyperaccumulator Sedum alfredii Hance and the fast-growing, high-biomass willow (Salix sp.). We investigated the efficiency of AC field combined with S. alfredii-willow intercropping for removing Cd from soils with different pH values. In the AC electric field treatment with S. alfredii-willow intercropping, the available Cd content in acidic soil increased by 50.00% compared to the control, and in alkaline soil, the increase was 100.00%. Furthermore, AC electric field promoted Cd uptake by plants in both acidic and alkaline soils, with Cd accumulation in the aboveground increased by 20.52% (P < 0.05) and 11.73%, respectively. In conclusion, the integration of AC electric fields with phytoremediation demonstrates significant favorable effectiveness., (© 2024. The Author(s).)

Most hyperaccumulators cannot maintain vigorous growth throughout the year, which may result in a low phytoextraction efficiency for a few months. In the present study, rotation of two hyperaccumulators is proposed to address this issue. An 18-month field experiment was conducted to evaluate the phytoextraction efficiency of Cd by the monoculture and rotation of Celosia argentea and Sedum plumbizincicola. The results showed that rotation increased amount of extracted Cd increased by 2.3 and 1.6 times compared with monoculture of C. argentea and S. plumbizincicola. In rotation system, the biomass of S. plumbizincicola and Cd accumulation in C. argentea increased by 54.4% and 40.7%, respectively. Rotation reduced fallow time and increased harvesting frequency, thereby enhancing Cd phytoextraction. Planting C. argentea significantly decreased soil pathogenic microbes and increased the abundances of plant growth-promoting rhizobacteria (PGPR) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes, which may be beneficial for the growth of S. plumbizincicola. Planting S. plumbizincicola increased the abundance of sulfur oxidization (SOX) system genes and decreased soil pH (p < 0.05), thereby increasing the Cd uptake by C. argentea. These findings indicated that rotation of C. argentea and S. plumbizincicola is a promising method for promoting Cd 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. Published by Elsevier B.V.)

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.)

Plant growth regulators (PGR) and plant growth-promoting bacteria (PGPB) have the potential in phytoremediation of heavy metals (HMs) contaminated soils. However, their sole application may not yield the optimal results, thus necessitating the combined application. The present study aimed to enhance the phytoremediation efficiency of Sedum alfredii Hance (S. alfredii) in acidic and alkaline soils through the combination of PGR (Brassinolide, BR) and PGPB (Pseudomonas fluorescens, P. fluorescens). The combination of BR and P. fluorescens (BRB treatment) effectively increased the removal efficiency of S. alfredii for Cd, Pb, and Zn by 355.2 and 155.3 %, 470.1 and 128.9 %, and 408.4 and 209.6 %, in acidic and alkaline soils, respectively. Moreover, BRB treatment led to a substantial increase in photosynthetic pigments contents and antioxidant enzymes activities, resulting in a remarkable increase in biomass (86.71 and 47.22 %) and dry mass (101.49 and 42.29 %) of plants grown in acidic and alkaline soils, respectively. Similarly, BRB treatment significantly elevated the Cd (109.4 and 71.36 %), Pb (174.9 and 48.03 %), and Zn levels (142.8 and 104.3 %) in S. alfredii shoots, along with cumulative accumulation of Cd (122.7 and 79.47 %), Pb (183.8 and 60.49 %), and Zn (150.7 and 117.9 %), respectively. In addition, the BRB treatment lowered the soil pH and DTPA-HMs contents, while augmenting soil enzymatic activities, thereby contributing soil microecology and facilitating the HMs absorption and translocation by S. alfredii to over-ground tissues. Furthermore, the evaluation of microbial community structure in phyllosphere and rhizosphere after remediation revealed the shift in microbial abundance. The combined treatment altered the principal effects on S. alfredii HMs accumulation from bacterial diversity to the soil HMs availability. In summary, our findings demonstrated that synergistic application of BR and P. fluorescens represents a viable approach to strengthen the phytoextraction efficacy of S. alfredii in varying soils., 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.)

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.)

Phytoremediation coupled with agroproduction (PCA) model contributes to sustainable agriculture and environmental management. This study investigated the impact of continuous cropping early/late season rice (RR) and Sedum alfredii-rice rotation (SR) on soil physical and chemical properties, as well as their relationships with soil microbial community. In 2022, SR treatment significantly increased pH value and organic matter content by 7 % and 17 %, respectively, compared to the levels in 2020, while RR treatment showed no change. RR treatment resulted in a significant decrease in soil concentrations of Ca, Mg, and K by 18.42 %, 29.01 %, and 7.77 %, respectively. Furthermore, SR treatment saw reductions of 29.62 % in total Cd and 38.30 % in DTPA extractable Cd in the soil. Over the two years, both treatments notably influenced the diversity, structure, and network of the rhizosphere bacterial and fungal communities, which are crucial for nutrient cycling and plant health. Notably, SR treatment exhibited a more complex network compared to RR, suggesting a greater impact on the interconnected systems. Therefore, these findings highlight the potential of Sedum rotation system to rehabilitate contaminated soils while supporting agricultural practices, which is essential for food security and environmental sustainability. This research direction holds promise for future exploration and application in the fields of phytoremediation and agroecology., 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. Published by Elsevier B.V.)

The focus on phytoremediation in soil cadmium (Cd) remediation is driven by its cost-effectiveness and eco-friendliness. Selecting suitable hyperaccumulators and optimizing their growth conditions are key to enhance the efficiency of heavy metal absorption and accumulation. Our research has concentrated on the role of salicylic acid (SA) and jasmonic acid (JA) in facilitating Cd phytoextraction by "Sedum alfredii (S. alfredii)" through improved soil-microbe interactions. Results showed that SA or JA significantly boosted the growth, stress resistance, and Cd extraction efficiency in S. alfredii. Moreover, these phytohormones enhanced the chemical and biochemical attributes of the rhizosphere soil, such as pH and enzyme activity, affecting soil-root interactions. High-throughput sequencing analysis has shown that Patescibacteria and Umbelopsis enhanced S. alfredii's growth and Cd extraction by modifying the bioavailability and the chemical conditions of Cd in soil. Structural Equation Model analysis further verified that phytohormones significantly enhanced the interaction between S. alfredii, soil, and microbes, leading to a marked increase in Cd accumulation in the plant. These discoveries emphasized the pivotal role of phytohormones in modulating the hyperaccumulators' response to environmental stress and offered significant scientific support for further enhancing the potential of hyperaccumulators in ecological restoration technologies using phytohormones., 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.)

Soil cadmium (Cd) pollution is escalating, necessitating effective remediation strategies. This study investigated the effects of exogenous jasmonic acid (JA) on Sedum alfredii Hance under Cd stress, aiming to enhance its phytoextraction efficiency. Initially, experiments were conducted to assess the impact of various concentrations of JA added to environments with Cd concentrations of 100, 300, and 500 μmol/L. The results determined that a concentration of 1 μmol/L JA was optimal. This concentration effectively mitigated the level of ROS products by enhancing the activity of antioxidant enzymes. Additionally, JA fostered Cd absorption and accumulation, while markedly improving plant biomass and photosynthetic performance. In further experiments, treatment with 1 μmol/L JA under 300 μmol/L Cd stress was performed and transcriptomic analysis unveiled a series of differentially expressed genes (DEGs) instrumental in the JA-mediated Cd stress response. These DEGs encompass not only pathways of JA biosynthesis and signaling but also genes encoding functions that influence antioxidant systems and photosynthesis, alongside genes pertinent to cell wall synthesis, and metal chelation and transport. This study highlights that JA treatment significantly enhances S. alfredii's Cd tolerance and accumulation, offering a promising strategy for plant remediation and deepening our understanding of plant responses to heavy metal stress., 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.)

Sedum plumbizincicola is a promising hyperaccumulator for heavy metal phytoremediation. It grows in heavy metal polluted soil and stores specific endophyte resources with heavy metal tolerance or growth promotion characteristics. In this study, the endophyte communities of S. plumbizincicola, growing naturally in the field (two former mining locations and one natural location) were investigated, and their structure and function were comparatively studied. The bioaccumulation and translocation characteristics of cadmium (Cd) and selenium (Se) in S. plumbizincicola were also evaluated. The results showed that the heavy metal pollution reduced the richness and diversity of endophyte communities. Soil pH and Cd concentration could be the key factors affecting the composition of the endophyte community. Co-occurrence network analysis identified that 22 keystone taxa belonging to Actinobacteriota, Firmicutes, Myxococcota and Proteobacteria were positively correlated with Cd bioaccumulation and translocation. The predicted endophyte metabolic pathways were enriched in physiological metabolism, immune system, and genetic Information processing. These findings may help to understand how endophytes assist host plants to enhance their adaptability to harsh environments, and provide a basis for further exploration of plant-endophyte interactions and improvement in phytoremediation efficiency., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Cd tolerance protein SaCTP3, which responds to Cd stress, was identified in Sedum alfredii; however, how to improve the efficiency of phytoremediation of Cd-contaminated soil using the CTP gene remains unknown. In this study, the phytoremediation potential of SaCTP3 of Sedum alfredii was identified. In the yeast Cd-sensitive strain Δycf1 overexpressing SaCTP3, the accumulation of Cd was higher than that in the Δycf1 strain overexpressing an empty vector. Transgenic sorghum plants overexpression SaCTP3 were further constructed to verify the function of SaCTP3. Compared to wild-type plants, the SaCTP3-overexpressing lines exhibited higher Cd accumulation under 500 μM Cd conditions. The average Cd content inSaCTP3-overexpressing plants is more than four times higher than that of WT plants. This was accompanied by an enhanced ability to scavenge ROS, as evidenced by the significantly increased activities of peroxidase, catalase, and superoxide dismutase in response to Cd stress. Pot experiments further demonstrated that SaCTP3 overexpression resulted in improved soil Cd scavenging and photosynthetic abilities. After 20 days of growth, the average Cd content in the soil planted with SaCTP3-overexpressing sorghum decreased by 19.4%, while the residual Cd content in the soil planted with wild-type plants was only reduced by 5.4%. This study elucidated the role of SaCTP3 from S.alfredii, highlighting its potential utility in genetically modifying sorghum for the effective phytoremediation of Cd., 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.)

The selection of appropriate plants and growth strategies is a key factor in improving the efficiency and universal applicability of phytoremediation. Sedum lineare grows rapidly and tolerates multiple adversities. The effects of inoculation of Acinetobacter sp. phosphate solubilizing bacteria P-1 and application of phosphate rock (PR) as additives on the remediation efficiency of As-contaminated soil by S. lineare were investigated. Compared with the control, both the single treatment and the combination of inoculation with strain P-1 and application of PR improved the biomass by 30.7-395.5%, chlorophyll content by 48.1-134.8%, total protein content by 12.5-92.4% and total As accumulation by 45.1-177.5%, and reduced the As-induced oxidative damage. Inoculation with strain P-1 increased the activities of superoxide dismutases and catalases of S. lineare under As stress, decreased the accumulation of reactive oxygen species in plant tissues and promoted the accumulation of As in roots. In contrast, simultaneous application of PR decreased As concentration in S. lineare tissues, attenuated As-induced lipid peroxidation and improved As transport to shoots. In addition, the combined application showed the best performance in improving resistance and biomass, which significantly increased root length by 149.1%, shoot length by 33%, fresh weight by 395.5% and total arsenic accumulation by 159.2%, but decreased the malondialdehyde content by 89.1%. Our results indicate that the combined application of strain P-1 and PR with S. lineare is a promising bioremediation strategy to accelerate phytoremediation of As-contaminated soils., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Pollution accident of nonferrous metallurgy industry often lead to serious heavy metal pollution of the surrounding soil. Phytoremediation of contaminated soil is an environmental and sustainable technology, and soil native microorganisms in the process of phytoremediation also participate in the remediation of heavy metals. However, the effects of high concentrations of multiple heavy metals (HCMHMs) on plants and native soil microorganisms remain uncertain. Thus, further clarification of the mechanism of phytoremediation of HCMHMs soil by plants and native soil microorganisms is required. Using the plant Sedum alfredii (S. alfredii) to restore HCMHM-contaminated soil, we further explored the mechanism of S. alfredii and native soil microorganisms in the remediation of HCMHM soils. The results showed that (i) S. alfredii can promote heavy metals from non-rhizosphere soil to rhizosphere soil, which is conducive to the effect of plants on heavy metals. In addition, it can also enrich the absorbed heavy metals in its roots and leaves; (ii) native soil bacteria can increase the abundance of signal molecule-synthesizing enzymes, such as trpE, trpG, bjaI, rpfF, ACSL, and yidC, and promote the expression of the pathway that converts serine to cysteine, then synthesize substances to chelate heavy metals. In addition, we speculated that genes such as K19703, K07891, K09711, K19703, K07891, and K09711 in native bacteria may be involved in the stabilization or absorption of heavy metals. The results provide scientific basis for S. alfredii to remediate heavy metals contaminated soils, and confirm the potential of phytoremediation of HCMHM contaminated soil., 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 © 2023. Published by Elsevier B.V.)

Intercropping of specific accumulators with industrial crops is used in moderately metal contaminated agricultural land. The distribution characteristics and environmental risks of non-accumulated ions in intercropping fields have rarely been reported. This study analyzed dissolved organic matter (DOM) fractionation and metal chemical forms to investigate the bioavailability, transformation, and uptake of non-hyperaccumulated metals in different cultivation patterns of a Cd hyperaccumulator (Sedum alfredii Hance) and a commercial crop (Cicer arietinum L.). The study focused on the distribution and transformation of heavy metals, with a particular emphasis on the role of DOM in intercropping. The contents of DOM in the rhizosphere soils of the Cd hyperaccumulator monoculture and the intercropping field were obviously greater than the DOM concentration in the commercial crop monoculture. The content of soluble Cd was significantly lower in the former two planting patterns than in the latter. In contrast, soluble Pb and Cu exhibited opposite content characteristics. In addition, the metal extraction ability of DOM extracted from the C. arietinum monoculture was lower than those from the Cd hyperaccumulator monoculture and the intercropping field. The concentrations of Cd in both below-ground and aerial parts of C. arietinum intercropping were significantly lower than those in its monoculture, since S. alfredii depleted soil Cd. Contrastingly, the contents of Cu and Pb in C. arietinum harvested from intercropping were significantly greater than those in its monoculture because the intercropped Cd hyperaccumulator activated Cu and Pb by changing soil DOM content and fractionations without absorbing them. The findings provide valuable insights into the use of intercropping to remediate moderately metal-contaminated agricultural land and highlight the potential risks associated with intercropping in multi-metal-contaminated fields., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Modeling plants for biomass production and metal uptake from surrounding environment is strongly dependent on the moisture content of soil. Therefore, experiments were conducted to find out how soil moisture affects the phenotypic traits, photosynthetic efficiency, metabolic profile, and metal accumulation in the hyperaccumulating ecotype of Sedum alfredii (S. alfredii). A total of six water potential gradients were set: 0 ~ -15 kPa (T1), -15 ~ -30 kPa (T2), -30 ~ -45 kPa (T3), -45 ~ -60 kPa (T4), -60 ~ -75 kPa (T5), and -75 ~ -90 kPa (T6). Different water potential treatments had a significant effect on plant growth and metal uptake efficiency. Compared to T3, T2 was more effective in promoting plant growth and development, with an increase in biomass of 23% and 17% in both fresh weight (FW) and dry weight (DW), respectively. T2 and T3 had the highest cadmium (Cd) content in the shoot (280.2 mg/kg) and (283.3 mg/kg), respectively, whereas T1 had the lowest values (204.7 mg/kg). Cd availability for plants in the soil was affected by moving soil moisture cycles. Changes in soil moisture that were either too high or too low compared to the ideal soil water content for S. alfredii growth resulted in a significant reduction in Cd accumulation in shoots. Tryptophan, phenylalanine, and other amino acids were accumulated in T5, whereas only tryptophan and phenylalanine slightly increased in T1. Sugars and alcohols such as sucrose, trehalose, mannitol, galactinol, and mannobiose increased in T5, while they decreased significantly in T1. Interestingly, in contrast to T1, the two impaired metabolic pathways in T5 (galactose and starch metabolism) were identified to be glucose metabolic pathways. These findings provide scientific information (based on experiments) to improve biomass production and metal uptake efficiency in hyperaccumulating ecotype of S. alfredii for phytoremediation-contaminated agricultural fields., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The cadmium hyperaccumulator Sedum plumbizincicola has remarkable abilities for cadmium (Cd) transport, accumulation and detoxification, but the transcriptional regulation mechanisms responsible for its Cd hyperaccumulation remain unknown. To address this knowledge gap, we conducted a comparative transcriptome study between S. plumbizincicola and the non-hyperaccumulating ecotype (NHE) of Sedum alfredii with or without Cd treatment. Our results revealed many differentially expressed genes involved in heavy metal transport and detoxification that were abundantly expressed in S. plumbizincicola . Additionally, we identified a large number of differentially expressed transcription factor genes, highlighting the complexity of transcriptional regulatory networks. We further screened four transcription factor genes that were highly expressed in the roots of S. plumbizincicola as candidate genes for creating CRISPR/Cas9 knockout mutations. Among these, the SpARR11 and SpMYB84 mutant lines exhibited decreased Cd accumulation in their aboveground parts, suggesting that these two transcription factors may play a role in the regulation of the Cd hyperaccumulation in S. plumbizincicola . Although further research will be required to determine the precise targeted genes of these transcription factors, combined transcriptome analysis and CRISPR/Cas9 technology provides unprecedented opportunities for identifying transcription factors related to Cd hyperaccumulation and contributes to the understanding of the transcriptional regulation mechanism of hyperaccumulation in S. plumbizincicola .

Cd contamination is a world-wild concern for its toxicity and accumulation in food chain. Sedum alfredii Hance (Crassulaceae) is a zinc (Zn) and cadmium (Cd) hyperaccumulator native to China and widely applied for the phytoremediation at Zn or Cd contaminated sites. Although many studies report the uptake, translocation and storage of Cd in S. alfredii Hance, limited information is known about the genes and underlying mechanisms of genome stability maintenance under Cd stress. In this study, a gene resembling DNA-damage repair/toleration 100 (DRT100) was Cd inducible and designated as SaDRT100. Heterologous expression of SaDRT100 gene in yeasts and Arabidopsis thaliana enhanced Cd tolerance capability. Under Cd stress, transgenic Arabidopsis with SaDRT100 gene exhibited lower levels of reactive oxygen species (ROS), fewer Cd uptake in roots and less Cd-induced DNA damage. Evidenced by the subcellular location in cellular nucleus and expression in aerial parts, we suggested the involvement of SaDRT100 in combating Cd-induced DNA damage. Our findings firstly uncovered the roles of SaDRT100 gene in Cd hypertolerance and genome stability maintenance in S. alfredii Hance. The potential functions of DNA protection make SaDRT100 gene a candidate in genetic engineering for phytoremediation at multi-component contaminated sites., 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 © 2023 Elsevier Ltd. All rights reserved.)

Phytoextraction is an environmentally friendly phytoremediation technology that can reduce the total amount of heavy metals (HMs) in the soil. Hyperaccumulators or hyperaccumulating transgenic plants with biomass are important biomaterials for phytoextraction. In this study, we show that three different HM transporters from the hyperaccumulator Sedum pumbizincicola, SpHMA2, SpHMA3, and SpNramp6, possess Cd transport. These three transporters are located at the plasma membrane, tonoplast, and plasma membrane, respectively. Their transcripts could be strongly stimulated by multiple HMs treatments. To create potential biomaterials for phytoextraction, we overexpressed the three single genes and two combining genes, SpHMA2&SpHMA3 and SpHMA2&SpNramp6, in rapes having high biomass and environmental adaptability, and found that the aerial parts of the SpHMA2-OE3 and SpHMA2&SpNramp6-OE4 lines accumulated more Cd from single Cd-contaminated soil because SpNramp6 transports Cd from root cells to the xylem and SpHMA2 from the stems to the leaves. However, the accumulation of each HM in the aerial parts of all selected transgenic rapes was strengthened in multiple HMs-contaminated soils, probably due to the synergistic transport. The HMs residuals in the soil after the transgenic plant phytoremediation were also greatly reduced. These results provide effective solutions for phytoextraction in both Cd and multiple HMs-contaminated soils., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yan-Rong Luo, Kun-Ming Chen has patent #CN108384802A issued to Guangdong Kaiyuan Environmental Technology Co., Ltd. Kun-Ming Chen has patent # CN110527694A issued to Guangdong Kaiyuan Environmental Technology Co., Ltd; Northwest A&F University., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) pollution is a global ecological soil problem. Screening and establishing an efficient phytoremediation system would be beneficial for alleviating this problem. The ornamental plant Sedum spectabile was selected as the remediation plant to study the removal efficiencies of PAHs after adding different concentrations of nano-SiO 2 , nano-CeO 2 , and traditional Na-montmorillonite (Na-MMT). The results demonstrated that shoot biomass was increased and photosynthesis was enhanced by the nanomaterial amendments. The uptake of 16 PAHs by S. spectabile was remarkably increased. Moreover, the two highest shoot concentrations were 7.61 (Phe) and 12.03 (Flo) times that of the control, and the two highest translocation factors were 31 (BbF) and 28 (BaP) times that of the control. Furthermore, 16S rRNA gene sequencing showed that the addition of nano-SiO 2 increased the abundance of Acidobacteria, and the genera related to PAH degradation was higher under nanomaterial treatments. The very high Si concentration in the shoots of S. spectabile had a significant linear correlation with the concentration of PAHs. In conclusion, the S. spectabile remediation system assisted by two nanomaterials was effective for the removal of PAHs from soil, and the transfer of PAHs to easily harvested aboveground plant parts was especially worthy of attention., 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 © 2023 Elsevier B.V. All rights reserved.)

This study employs stable isotope analysis to investigate the mechanisms of cadmium (Cd) and zinc (Zn) interaction in the metal hyperaccumulating plant species Sedum plumbizincicola . To this end, the Cd and Zn isotope compositions of root, stem, leaf, and xylem sap samples were determined during metal uptake and translocation at different Cd and Zn concentrations. The enrichment of light isotopes of both elements in plants during uptake was less pronounced at low metal supply levels, likely reflecting the switch from a low-affinity to a high-affinity transport system at lower levels of external metal supply. The lower δ 114/110 Cd values of xylem sap when treated with a metabolic inhibitor decreasing the active Cd uptake further supports the preference of heavier Cd isotopes during high-affinity transport. The Δ 66 Zn plant-initial solution or Δ 66 Zn plant-final solution values were similar at different Cd concentrations, indicating negligible interaction of Cd in the Zn uptake process. However, decreasing Zn supply levels significantly increased the enrichment of light Cd isotopes in plants (Δ 114/110 Cd = -0.08‰) in low-Cd treatments but reduced the enrichment of light Cd isotopes in plants (Δ 114/110 Cd = 0.08‰) under high Cd conditions. A systematic enrichment of heavy Cd and light Zn isotopes was found in root-to-shoot translocation of the metals. The Cd concentrations of the growth solutions thereby had no significant impact on Zn isotope fractionation during root-to-shoot translocation. However, the Δ 114/110 Cd translocation values hint at possible competition between Cd and Zn for transporters during root-to-shoot transfer and this may impact the transport pathway of Cd. The stable isotope data demonstrate that the interactions between the two metals influenced the uptake and transport mechanisms of Cd in S. plumbizincicola but had little effect on those of Zn.

Hyperaccumulators are plant species that tolerate and accumulate very high concentrations of toxic metals, including Cd. Hyperaccumulation of heavy metals is reported to benefit plant biotic resistance; however, no prior study has examined the possible role of toxic metals on abiotic stress resistance in hyperaccumulators. A preliminary experiment found that Cd significantly improved plant growth of a hyperaccumulator, Sedum alfredii Hance, under heat stress. This study investigated the possible role of Cd in S. alfredii's heat resistance, using infrared thermography, transmission electron microscopy (TEM), real-time quantitative polymerase chain reaction (RTqPCR), and high-throughput sequencing. The results showed that high temperatures irreversibly damaged stomatal function, chloroplast structure, photosynthesis in S. alfredii, and lowered survival rates to 25%. However, Cd application significantly decreased the leaf temperature of S. alfredii and increased the survival rate to 75%. Cd penetrated the guard cells, restored stomatal function, and mitigated excessive water loss from S. alfredii under heat stress. Moreover, it activated antioxidant enzymes, promoted phytohormone biosynthesis, and upregulated a series of unigenes, thereby augmenting heat resistance in S. alfredii. These results indicate that Cd effectively improved thermotolerance in S. alfredii by regulating stomatal movement and antioxidant systems via upregulation of phytohormones and heat shock proteins., 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 © 2022 Elsevier B.V. All rights reserved.)

Metallothioneins (MTs), a group of cysteine-rich proteins, are effective chelators of cadmium (Cd) and play a key role in plant Cd detoxification. However, little is known about the role of single cysteine (Cys) residues in the MTs involved in the adaptation of plants to Cd stress, especially, in hyperaccumulators. In the present study, we functionally characterised SaMT3 in S. alfredii, a Cd/Zn hyperaccumulator native to China. Our results showed that the C- and N- terminal regions of SaMT3 had differential functional natures in S. alfredii and determined its Cd hypertolerance and detoxification. Two CXC motifs within the C-terminal region were revealed to play a crucial role in Cd sensing and binding, whereas the four Cys-residues within the N-terminal region were involved in scavenging reactive oxygen species (ROS). An S. alfredii transgenic system based on callus transformation was developed to further investigate the in-planta gene function. The SaMT3-overexpressing transgenic plant roots were more tolerant to Cd than those of wild-type plants. Knockout of SaMT3 resulted in significantly decreased Cd concentrations and increased ROS levels after exposure to Cd stress. We demonstrated the SaMT3-mediated adaptation strategy in S. alfredii, which uses metal chelation and ROS scavenging in response to Cd stress. Our results further reveal the molecular mechanisms underlying Cd detoxification in hyperaccumulating plants, as well as the relation between Cys-related motifs and the metal binding properties of MTs. This research provides valuable insights into the functions of SaMT3 in S. alfredii, and improves our understanding of Cd hyperaccumulation in 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 © 2022 Elsevier Ltd. All rights reserved.)

Botrytis cinerea is a highly destructive and widespread phytopathogen in fruits. The widespread use of chemical antifungal agents on fruits has aided in disease control while their long-term use has resulted in the emergence of resistant fungal strains. Flavonoids have a specific antifungal effect. The inhibitory effect and underlying mechanism of flavonoids from Sedum aizoon L. (FSAL) on B. cinerea were determined in this study. The results showed that the minimum inhibitory concentration of FSAL against B. cinerea was 1.500 mg/mL. FSAL treatment caused leakage of macromolecules such as nucleic acids, led to accumulation of malondialdehyde and relative oxygen species, and disrupted the ultrastructure of B. cinerea. The transcriptome results indicated that compared with the control group, there were 782 and 1330 genes identified as being substantially upregulated and downregulated, respectively, in the FSAL-treated group. The identified genes and metabolites were mostly involved in redox processes and glycerolipid and amino acid metabolism pathways. FSAL offer a promising choice for food prevention and safety. KEY POINTS: • FSAL negatively affects the glycerolipid metabolism of B. cinerea • FSAL minimum inhibitory concentration against B. cinerea was 1.500 mg/mL • FSAL could be utilized as a new prevention strategy for gray mold in fruits., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitric oxide (NO) and ethylene are both important signaling molecules which participate in numerous plant development processes and environmental stress resistance. Here, we investigate whether and how NO interacts with ethylene during the development of endodermal barriers that have major consequences for the apoplastic uptake of cadmium (Cd) in the hyperaccumulator Sedum alfredii. In response to Cd, an increased NO accumulation, while a decrease in ethylene production was observed in the roots of S. alfredii. Exogenous supplementation of NO donor SNP (sodium nitroprusside) decreased the ethylene production in roots, while NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) had the opposite effect. The exogenous addition of NO affected the ethylene production through regulating the expression of genes related to ethylene synthesis. However, upon exogenous ethylene addition, roots retained their NO accumulation. The abovementioned results suggest that ethylene is downstream of the NO signaling pathway in S. alfredii. Regardless of Cd, addition of SNP promoted the deposition of endodermal barriers via regulating the genes related to Casparian strips deposition and suberization. Correlation analyses indicate that NO positively modifies the formation of endodermal barriers via the NO-ethylene signaling pathway, Cd-induced NO accumulation interferes with the synthesis of ethylene, leading to a deposition of endodermal barriers in S. alfredii., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Sedum alfredii Hance is a perennial herb native to China that can particularly be found in regions with abandoned Pb/Zn mines. It is a Cd/Zn hyperaccumulator that is highly tolerant to Pb, Cu, Ni, and Mn, showing potential for phytoremediation of soils contaminated with multiple heavy metals. A better understanding of how this species responds to different heavy metals would advance the phytoremediation efficiency. In this study, transcriptomic regulation of S. alfredii roots after Cd, Zn, Pb, and Cu exposure was analyzed to explore the candidate genes involved in multi-heavy metal tolerance. Although Zn and Cd, Pb and Cu had similar distribution patterns in S. alfredii, distinct expression patterns were exhibited among these four metal treatments, especially about half of the differentially expressed genes were upregulated under Cu treatment, suggesting that it utilizes distinctive and flexible strategies to cope with specific metal stress. Most unigenes regulated by Cu were enriched in catalytic activity, whereas the majority of unigenes regulated by Pb had unknown functions, implying that S. alfredii may have a unique strategy coping with Pb stress different from previous cognition. The unigenes that were co-regulated by multiple heavy metals exhibited functions of antioxidant substances, antioxidant enzymes, transporters, transcription factors, and cell wall components. These metal-induced responses at the transcriptional level in S. alfredii were highly consistent with those at the physiological level. Some of these genes have been confirmed to be related to heavy metal absorption and detoxification, and some were found to be related to heavy metal tolerance for the first time in this study, like Metacaspase-1 and EDR6. These results provide a theoretical basis for the use of genetic engineering technology to modify plants by enhancing multi-metal tolerance to promote phytoremediation efficiency., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Soil Cd and Zn contamination has become a serious environmental problem. This work explored the performance of wood vinegar (WV) in enhancing the phytoextraction of Cd/Zn by hyperaccumulator Sedum alfredii Hance. Rhizosphere chemical properties, enzyme activities and bacterial community were analyzed to determine the mechanisms of metal accumulation in this process. Results demonstrated that, after 120 days growth, different times dilution of WV increased the shoot biomass of S. alfredii by 85.2%-148%. In addition, WV application significantly increased soil available Cd and Zn by lowing soil pH, which facilitated plant uptake. The optimal Cd and Zn phytoextraction occurred from the 100 times diluted WV (D100), which increased the Cd and Zn extraction by 188% and 164%, compared to CK. The 100 and 50 times diluted WV significantly increased soil total and available carbon, nitrogen and phosphorus, and enhancing enzyme activities of urease, acid phosphatase, invertase and protease by 10.1-21.4%, 29.1-42.7%,12.2-38.3% and 26.8-85.7%, respectively, compared to CK. High-throughput sequencing revealed that the D 100 significantly increased the bacterial diversity compared to CK. Soil bacterial compositions at phylum, family and genera level were changed by WV addition. Compared to CK, WV application increased the relative abundances of genus with plant growth promotion and metal mobilization function such as, Bacillus, Gemmatimonas, Streptomyces, Sphingomonas and Polycyclovorans, which was positively correlated to biomass, Cd/Zn concentrations and extractions by S. alfredii. Structural equation modeling analysis showed that, soil chemical properties, enzyme activities and bacterial abundance directly or indirectly contributed to the biomass promotion, Cd, and Zn extraction by S. alfredii. To sum up, WV improved phytoextraction efficiency by enhancing plant growth, Cd and Zn extraction and increasing soil nutrients, enzyme activities, and modifying bacterial community., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Soil collembolans live in close proximity to plant roots and may have a role in the phytoextraction of potentially toxic metals from contaminated soils but the underlying mechanisms remain poorly investigated. We hypothesize that soil collembolans may change the root morphology of hyperaccumulators by regulating plant physiological characteristics. Here, a pot experiment was conducted in which a cadmium (Cd) and zinc (Zn) hyperaccumulator (Sedum plumbizincicola) was grown with or without a collembolan (Folsomia candida), and plant transcriptome and hormones as well as the root characteristics of S. plumbizincicola were analyzed. F. candida promoted the growth and Cd/Zn uptake of S. plumbizincicola, the root and shoot biomass increasing by 53.3 and 34.4%, and the uptake of Cd and Zn in roots increased by 83.2 and 65.4%, respectively. Plant root morphology, total root length, root tip number and lateral root number increased significantly by 40.7, 37.2 and 33.8%, respectively, with the addition of F. candida. Transcriptome analysis reveals that the expression levels of defense-related genes in S. plumbizincicola were significantly up-regulated. In addition, the defensive plant hormones, i.e. salicylic acid in the roots, increased significantly by 338%. These results suggest that the plant in defense of the action of F. candida regulated the expression of the corresponding genes and increased the defensive plant hormones, thus modifying root morphology and plant performance. Overall, this study highlights the importance of the regulation by collembolans of plant growth and metal uptake by interaction with hyperaccumulator roots., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The interactions between hyperaccumulators and their associated herbivores have been mostly investigated in their natural habitats and largely ignored in the phytoremediation practice. Herein, we investigated the herbivory status of Zn/Cd-hyperaccumulating plant Sedum alfredii from both their natural habitats and their applied remediation field, and inspected the adaptive strategies of the herbivores from the perspective of their facilitative gut microbiota. Field investigations showed that snail species Bradybaena ravida was the dominant herbivore feeding on S. alfredii and they can be only found in sites with lower levels of heavy metals compared with the plant natural habitat. Gut microbial community was analyzed using two sequencing methods (16S rRNA and czcA-Zn/Cd resistant gene) to comparatively understand the effect of gut microbes in facilitating snail feeding on the hyperaccumulators. The results revealed significant differences in the diversity and richness between the gut microbiota of the two snail populations, which was more pronounced by the czcA sequencing method. Despite of the compositional differences, their functions seemed to converge into three categories as metal-tolerant and contaminant degraders, gut symbionts, and pathogens. Further function potentials predicted by Tax4Fun based on 16 S sequencing data were in accordance with this categorization as the most abundant metabolic pathways were two-component system and ABC transporter, which was closely related to metal stress adaptation. The prevalence of positive interactions (~80%) indicated by the co-occurrence network analysis based on czcA sequencing data in both groups of gut microbiota further suggested the facilitative effect of these metal-tolerant gut microbes in coping with the high metal diet, which ultimately assist the snails to successfully feed on S. alfredii plants and thrive. This work for the first time provides evidence that the herbivore adaptation to hyperaccumulators were also associated with their gut microbial adaptation to metals., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Sedum plumbizincicola is an herbaceous species tolerant of excessive cadmium accumulation in above-ground tissues. The implications of membrane proteins, especially integrative membrane proteins, in Cd detoxification of plants have received attention in recent years, but a comprehensive profiling of Cd-responsive membrane proteins from Cd hyperaccumulator plants is lacking. In this study, the membrane proteins of root, stem, and leaf tissues of S. plumbizincicola seedlings treated with Cd solution for 0, 1 or 4 days were analyzed by Tandem Mass Tag (TMT) labeling-based proteome quantification (Data are available via ProteomeXchange with identifier PXD025302). Total 3353 proteins with predicted transmembrane helices were identified and quantified in at least one tissue group. 1667 proteins were defined as DAPs (differentially abundant proteins) using fold change >1.5 with p-values <0.05. The number of DAPs involved in metabolism, transport protein, and signal transduction was significantly increased after exposure to Cd, suggesting that the synthesis and decomposition of organic compounds and the transport of ions were actively involved in the Cd tolerance process. The number of up-regulated transport proteins increased significantly from 1-day exposure to 4-day exposure, from 5 to 112, 16 to 42, 18 to 44, in root, stem, and leaf, respectively. Total 352 Cd-regulated transport proteins were identified, including ABC transporters, ion transport proteins, aquaporins, proton pumps, and organic transport proteins. Heterologous expression of SpABCB28, SpMTP5, SpNRAMP5, and SpHMA2 in yeast and subcellular localization showed the Cd-specific transport activity. The results will enhance our understanding of the molecular mechanism of Cd hypertolerance and hyperaccumulation in S. plumbizincicola and will be benefit for future genetic engineering in phytoremediation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

Main Conclusion: Cadmium-sensitive yeast screening resulted in the isolation of protein translation factor SaeIF1 from the hyperaccumulator Sedum alfredii which has both general and special regulatory roles in controlling cadmium accumulation. The hyperaccumulator of Sedum alfredii has the extraordinary ability to hyperaccumulate cadmium (Cd) in shoots. To investigate its underlying molecular mechanisms of Cd hyperaccumulation, a cDNA library was generated from leaf tissues of S. alfredii. SaeIF1, belonging to the eukaryotic protein translation factor SUI1 family, was identified by screening Cd-sensitive yeast transformants with this library. The full-length cDNA of SaeIF1 has 582 bp and encodes a predicted protein with 120 amino acids. Transient expression assays showed subcellular localization of SaeIF1 in the cytoplasm. SaeIF1 was constitutively and highly expressed in roots and shoots of the hyperaccumulator of S. alfredii, while its transcript levels showed over 100-fold higher expression in the hyperaccumulator of S. alfredii relative to the tissues of a nonhyperaccumulating ecotype of S. alfredii. However, the overexpression of SaeIF1 in yeast cells increased Cd accumulation, but conferred more Cd sensitivity. Transgenic Arabidopsis thaliana expressing SaeIF1 accumulated more Cd in roots and shoots without changes in the ratio of Cd content in shoots and roots, but were more sensitive to Cd stress than wild type. Both special and general roles of SaeIF1 in Cd uptake, transportation, and detoxification are discussed, and might be responsible for the hyperaccumulation characteristics of S. alfredii.

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.)

Plant growth-promoting bacteria (PGPB) can promote root uptake and shoot accumulation of cadmium (Cd) in hyperaccumulator plants, but the mechanisms by which PGPB accelerate root-to-shoot transport of Cd is still unknown. A better understanding of these mechanisms is necessary to develop the strategies that can promote the practical phytoextraction of Cd-polluted soils. In this study, we found that Pseudomonas fluorescens accelerates a reversed and a long-distance transport of Cd and sucrose in Sedum alfredii, by examining the xylem and phloem sap and by quantifying the concentrations of Cd and sucrose in shoot and root. The transcriptome sequencing has revealed the up-regulated expressions of starch metabolism and sucrose biosynthesis related genes in the shoots of Cd hyperaccumulator plant S. alfredii that was inoculated with PGPB P. fluorescens. In addition, the genes of sugar, cation and anion transporters were also up-regulated by bacterial treatment, showing a complicated co-expression network with sucrose biosynthesis related genes. The expression levels of Cd transporter genes, such as ZIP1, ZIP2, HMA2, HMA3 and CAX2, were elevated after PGPB inoculation. As a result, the PGPB successfully colonized the root, and promoted the sucrose shoot-to-root transport and Cd root-to-shoot transport in S. alfredii. Since non-photosynthetic root-associated bacteria usually obtain sugars from photosynthetic plants, our results highlight the importance of PGPB-induced changes in hyperaccumlator plants for both the host and the PGPB., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

SaNramp6 in Sedum alfredii encodes a membrane-localized metal transporter. We isolated the SaNramp6h allele from the hyperaccumulating ecotype (HE) of S. alfredii . When this allele was expressed in transgenic yeast and Arabidopsis thaliana , it enhanced their cadmium (Cd) sensitivity by increased Cd transport and accumulation. We isolated another allele, SaNramp6n , from a nonhyperaccumulating ecotype (NHE) of S. alfredii . Amino acid sequence comparisons revealed three amino acid differences between SaNramp6h and SaNramp6n. We investigated the Cd transport activity of the Nramp6 allele, and determined which residues are essential for the transport activity. We conducted structure-function analyses of SaNramp6 based on site-directed mutagenesis and functional assays of the mutants in yeast and Arabidopsis . The three residues that differed between SaNramp6h and SaNramp6n were mutated. Only the L157P mutation of SaNramp6h impaired Cd transport. The other mutations, S218N and T504A, did not affect the transport activity of SaNramp6h, indicating that these residues are not essential for metal selectivity. Transgenic plants overexpressing SaNramp6h L157P showed altered metal accumulation in shoots and roots. Our results suggest that the conserved site L157 is essential for the high metal transport activity of SaNramp6h. This information may be useful for limiting or increasing Cd transport by other plant natural resistance associated macrophage protein (NRAMP) proteins.

Pot-culture experiments were conducted to investigate the potential of microorganism-saponin assisted phytoremediation of cadmium (Cd) and benzo(a)pyrene (B[a]P) co-contaminated soil using Cd-hyperaccumulator Sedum alfredii. Results showed that B[a]P-degrading bacterium (Ochrobactrum intermedium B[a]P-16) inoculation significantly increased root (by 22.1-24.1%) and shoot (by 20.5-23.4%) biomass of S. alfredii, whereas the application of saponin had no effect on the growth of S. alfredii. The saponin solution at 2 g L -1 extracted more Cd and B[a]P than water, saponin enhanced Cd and B[a]P bioavailability in soil and thus promoted their uptake and accumulation in S. alfredii. The activity of B[a]P-16, dehydrogenase and polyphenol oxidase in co-contaminated soil was promoted by growing S. alfredii, and the application of B[a]P-16 and saponins caused a significant (P < 0.05) increase in both enzyme activities. The maximum B[a]P removal rate (82.0%) and Cd phytoextraction rate (19.5%) were obtained by co-application of S. alfredii with B[a]P-16 and saponin. The B[a]P-16 and plant promoted biodegradation were the predominant contributors towards removal of B[a]P from soil. A significant (P < 0.05) synergistic effect of B[a]P-16 and saponin on B[a]P and Cd removal efficiency was observed in this study. It is suggested that planting S. alfredii with application of B[a]P-16 and saponin would be an effective method for phytoremediation of soil co-contaminated with Cd and PAHs., (Copyright © 2019. Published by Elsevier Ltd.)

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.

Sedum plumbizincicola is a native cadmium/zinc (Cd/Zn) hyperaccumulator in China. At present, it has been applied to remediation of Cd contaminated soils. As the large differences in the removal efficiency of Cd and Zn for different soil conditions, the enhancement measure is important for the phytoremediation process. There have been many studies on the effects of earthworms on plant growth. But the effects on the growth of S. plumbizincicola and heavy metal removal efficiency have been rarely reported. There were 2 pot experiments: S. plumbizincicola was planted on 3 types of soils: Perudic Luvisols, Stagnic Anthrosols, and Udic Cambisols inoculated with Eisenia foetida to explore the effect of Eisenia foetida on the growth and Cd/Zn absorption of S. plumbizincicola. Stagnic Anthrosols with higher Cd effectiveness was selected in the second season pot experiment for further research the combined effect of earthworm and rich straw. The results of the first pot experiment showed that the addition of earthworms in acidic Perudic Luvisols increased the shoot biomass of S. plumbizincicola by 106% compared with the control treatment, and the Cd and Zn uptake increased by 72.0% and 36.0%, respectively. The soil available Cd was reduced by the addition of earthworms. The other two soils inoculated with earthworms had no enhancement on phytoremediation, addition of earthworms together with straw could improve the growth of S. plumbizincicola and the Cd/Zn uptake in Stagnic Anthrosols. The above results indicated that adding earthworm can enhance the phytoremediation of Cd/Zn hyperaccumulator, and the addition of straw is an important synergistic technique for earthworm-enhanced phytoremediation of S. plumbizincicola.

The root apex is the most active part for water and ions uptake, however, longitudinal alterations in root characteristics along root apex and consequences for metal uptake in hyperaccumulator are poorly understood. Here, we compared cadmium (Cd)-induced longitudinal alterations in root apex of two ecotypes of Sedum alfredii and assess their effects on Cd uptake. Under Cd treatment, cell death began from epidermis to the stele in non-hyperaccumulating ecotype (NHE) over time, and the number of dead cells was significantly higher than that in hyperaccumulating ecotype (HE). Cd-induced the presence of border-like cells (BLCs) surrounding the root tip of NHE prevented Cd from entering roots, however, almost no BLCs were observed in the root tip of in HE. Besides, Cd-treated NHE exhibited 76% and 52% decrease in the proportions of meristematic and elongation zone, respectively, resulting in lower Cd influx and less intensive Cd-fluorescence in these zones, as compared with HE. In the differentiation zone, Cd induced earlier initiation of root hairs (RHs), lower RHs-density, shorter RHs-length, thicker RHs-radius and less trichoblasts in NHE than those in HE. These remarkable variations led to less Cd influx and lower intensity of Cd-fluorescence in RHs of NHE than those of HE. Furthermore, decline in cell wall thickness under Cd exposure resulted in less cell-wall-bond Cd in the cell wall of HE. Therefore, Cd-induced alterations in root characteristics alongside root apex contributed to the difference in Cd uptake and accumulation between two ecotypes of S. alfredii., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sedum alfredii Hance is a zinc (Zn) and cadmium (Cd) hyperaccumulator plant. However, the regulatory role of plant hormones in the Zn or Cd uptake and accumulation of S. alfredii remains unclear. In this work, the growth, Cd accumulation, abscisic acid (ABA) synthesis and catabolism, malonaldehyde (MDA) content, and transcriptional level of some Cd stress response genes under ABA and Cd co-treatment were investigated to reveal the impact of ABA on Cd resistance and Cd accumulation of S. alfredii. The results show that 0.2 mg/L ABA and 100 μmol/L Cd co-treatment enhanced Cd accumulation and growth in S. alfredii, whereas lower or higher ABA concentrations weaken or even reverse this effect, which was positively correlated with endogenous ABA content. The increase in endogenous ABA content might be the results of the increasing ABA synthetase activities and decreasing ABA lytic enzyme, which was induced by the application of 0.2 mg/L ABA under 100 μmol/L Cd treatment. Principal component analysis (PCA) indicated that ABA impacted the expression pattern of Cd stress response genes, which coincided with the Cd accumulation pattern in the shoots of S. alfredii. Cross-over analysis of partial least squares-discriminant analysis (PLS-DA) and correlation analysis indicated that HsfA4c, HMA4 expression in roots, and HMA2, HMA3, CAD, NAS expression in shoots were correlated with endogenous ABA, which suggests that endogenous ABA improves Cd resistance of seedlings, switches the root-to-shoot transporter from HMA2 to HMA4, and transports more Cd into apoplasts to promote Cd accumulation in the shoots of S. alfredii. Taken together, ABA plays an essential role not only in Cd resistance but also in Cd transport from root to shoot in S. alfredii under Cd stress.

Sedum alfredii Hance is a cadmium (Cd)/zinc (Zn) hyperaccumulator native to China. However, its relatively low biomass restricted the large-scale application for heavy metal contamination remediation. The chromosome set doubling of S. alfredii in vitro was achieved by 0.1%-0.2% (W/V) colchicine treatment. The plant DNA ploidy was analyzed by flow cytometry and chromosome set doubling plants (CSD) were identified based on the obvious different sharp peak. A tissue culture experiment with different Cd treated levels and a field trial with natural polluted mined soil were conducted to study the effects of chromosome doubling on plant biomass and Cd accumulation in shoots. The results suggested that S. alfredii is a mixoploid. Compared with the wild type plants (WT), CSD exhibited typical "gigas" characteristics in morphology including stem thickness, root hair production, number of leaves and size of stoma guard cell. Fresh weight and dry weight of CSD were increased to 1.62-2.03-fold and 2.26-3.25-fold of WT. And Cd content of CSD showed a 17.49%-42.82% increase and 59% increase under tissue culture and field condition, accordingly. In addition, the TF and in BCF of CSD were 2.37- and 1.59-fold of WT, respectively. These results proved that it is feasible to promote phytoextraction efficiency of S. alfredii in Cd contaminated soils through chromosomal engineering, which provides a novel approach for hyperaccumulator application in phytoremediation., (Copyright © 2019. Published by Elsevier B.V.)

Biodegradable chelator-assisted phytoextraction is an effective method to enhance remediation efficiency of heavy metals. A greenhouse experiment was conducted to investigate the effects of S,S-ethylenediamine disuccinic acid (EDDS), citric acid (CA), and oxalic acid (OA) application before planting on the biomass and physiological characteristics of hyperaccumulator Sedum alfredii Hance, and its cadmium (Cd), lead (Pb), and zinc (Zn) uptake. The results showed that EDDS and CA slightly inhibited the plant growth, while the 1.0 mmol kg -1 (OA-1) and 2.5 mmol kg -1 OA (OA-2.5) addition produced 55.3% and 35.2% greater shoot biomass compared with the control, which may be related to that OA can produce higher leaf chlorophyll and soluble protein contents, as well as lower concentrations of malondialdehyde. At the same time, the concentrations of Pb and Zn in leaf after OA-2.5 treatment significantly increased by 127% and 28.4%, and the Cd, Pb, and Zn uptake by shoot was obviously enhanced by 21.5%, 117%, and 44.9% for OA-1 addition and by 39.1%, 80.0%, and 58.3% for OA-2.5 addition, respectively, in comparison with the control (P < 0.05). The reductions in available contents of Cd, Pb, and Zn in soil were observed after phytoextraction by Sedum alfredii Hance when OA was treated. These findings imply that OA was suitable for facilitating Sedum alfredii Hance to remove Cd, Pb, and Zn in co-contaminated soil.

Plants with facultative crassulacean acid metabolism (CAM) maximize performance through utilizing C3 or C4 photosynthesis under ideal conditions while temporally switching to CAM under water stress (drought). While genome-scale analyses of constitutive CAM plants suggest that time of day networks are shifted, or phased to the evening compared to C3, little is known for how the shift from C3 to CAM networks is modulated in drought induced CAM. Here we generate a draft genome for the drought-induced CAM-cycling species Sedum album. Through parallel sampling in well-watered (C3) and drought (CAM) conditions, we uncover a massive rewiring of time of day expression and a CAM and stress-specific network. The core circadian genes are expanded in S. album and under CAM induction, core clock genes either change phase or amplitude. While the core clock cis-elements are conserved in S. album, we uncover a set of novel CAM and stress specific cis-elements consistent with our finding of rewired co-expression networks. We identified shared elements between constitutive CAM and CAM-cycling species and expression patterns unique to CAM-cycling S. album. Together these results demonstrate that drought induced CAM-cycling photosynthesis evolved through the mobilization of a stress-specific, time of day network, and not solely the phasing of existing C3 networks. These results will inform efforts to engineer water use efficiency into crop plants for growth on marginal land., Competing Interests: The authors have declared that no competing interests exist.

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.)

Endophyte-assisted phytoremediation has gained increasing attention. However, the interacting mechanisms of endophytes and metal hyperaccumulators are still not clear. An endophytic bacterium Pseudomonas fluorescens Sasm05 inoculation promoted Sedum alfredii Hance rooting and root development, in which the specific root length (SRL) and average number of root tips (ART) increased to 2.09- and 3.35-fold, respectively. Sasm05 inoculation promoted plant growth, increased the chlorophyll content, and elevated Zn uptake of plant at excess Zn supply. At 200 μM Zn treatment level, Sasm05 inoculation increased plant biomass and the chlorophyll content by more than 40%, and root Zn content by 40%. Furthermore, Sasm05 inoculation upregulated the expression of the Zn transporter SaIRT1 to 3.43-fold in the roots, while another transporter SaNramp1 expression was increased to 38.66-fold in the roots and 7.53-fold in the shoots. Time course study showed the best effects of Sasm05 on plant biomass and the chlorophyll content were detected at 30 d, while for Zn content at 3 d. These results firstly provided molecular evidences of endophytic bacteria in facilitating host plant Zn uptake, which will absolutely benefit the understanding of interacting mechanisms between hyperaccumulators and their endophytes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Abscisic acid (ABA) is a key phytohormone underlying plant resistance to toxic metals. However, regulatory effects of ABA on apoplastic transport in roots and consequences for uptake of metal ions are poorly understood. Here, we demonstrate how ABA regulates development of apoplastic barriers in roots of two ecotypes of Sedum alfredii and assess effects on cadmium (Cd) uptake. Under Cd treatment, increased endogenous ABA level was detected in roots of nonhyperaccumulating ecotype (NHE) due to up-regulated expressions of ABA biosynthesis genes (SaABA2, SaNCED), but no change was observed in hyperaccumulating ecotype (HE). Simultaneously, endodermal Casparian strips (CSs) and suberin lamellae (SL) were deposited closer to root tips of NHE compared with HE. Interestingly, the vessel-to-CSs overlap was identified as an ABA-driven anatomical trait. Results of correlation analyses and exogenous applications of ABA/Abamine indicate that ABA regulates development of both types of apoplastic barriers through promoting activities of phenylalanine ammonialyase, peroxidase, and expressions of suberin-related genes (SaCYP86A1, SaGPAT5, and SaKCS20). Using scanning ion-selected electrode technique and PTS tracer confirmed that ABA-promoted deposition of CSs and SL significantly reduced Cd entrance into root stele. Therefore, maintenance of low ABA levels in HE minimized deposition of apoplastic barriers and allowed maximization of Cd uptake via apoplastic pathway., (© 2018 John Wiley & Sons Ltd.)

Sedum plumbizincicola is able to hyperaccumulate cadmium (Cd), a nonessential and highly toxic metal, in the above-ground tissues, but the mechanisms for its Cd hypertolerance are not fully understood. Here, we show that the heavy metal ATPase 1 (SpHMA1) of S. plumbizincicola plays an important role in chloroplast Cd detoxification. Compared with the HMA1 ortholog in the Cd nonhyperaccumulating ecotype of Sedum alfredii, the expression of SpHMA1 in the leaves of S. plumbizincicola was >200 times higher. Heterologous expression of SpHMA1 in Saccharomyces cerevisiae increased Cd sensitivity and Cd transport activity in the yeast cells. The SpHMA1 protein was localized to the chloroplast envelope. SpHMA1 RNA interference transgenic plants and CRISPR/Cas9-induced mutant lines showed significantly increased Cd accumulation in the chloroplasts compared with wild-type plants. Chlorophyll fluorescence imaging analysis revealed that the photosystem II of SpHMA1 knockdown and knockout lines suffered from a much higher degree of Cd toxicity than wild type. Taken together, these results suggest that SpHMA1 functions as a chloroplast Cd exporter and protects photosynthesis by preventing Cd accumulation in the chloroplast in S. plumbizincicola and hyperexpression of SpHMA1 is an important component contributing to Cd hypertolerance in S. plumbizincicola., (© 2018 John Wiley & Sons Ltd.)