SpbZIP60 confers cadmium tolerance by strengthening the root cell wall compartmentalization in Sedum plumbizincicola.

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.

SpSIZ1 from hyperaccumulator Sedum plumbizincicola orchestrates SpABI5 to fine-tune cadmium tolerance.

Sedum plumbizincicola is a renowned hyperaccumulator of cadmium (Cd), possesses significant potential for eco-friendly phytoremediation of soil contaminated with Cd. Nevertheless, comprehension of the mechanisms underpinning its Cd stress response remains constrained, primarily due to the absence of a comprehensive genome sequence and an established genetic transformation system. In this study, we successfully identified a novel protein that specifically responds to Cd stress through early comparative iTRAQ proteome and transcriptome analyses under Cd stress conditions. To further investigate its structure, we employed AlphaFold, a powerful tool for protein structure prediction, and found that this newly identified protein shares a similar structure with Arabidopsis AtSIZ1. Therefore, we named it Sedum plumbizincicola SIZ1 (SpSIZ1). Our study revealed that SpSIZ1 plays a crucial role in positively regulating Cd tolerance through its coordination with SpABI5. Overexpression of SpSIZ1 significantly enhanced plant resistance to Cd stress and reduced Cd accumulation. Expression pattern analysis revealed higher levels of SpSIZ1 expression in roots compared to stems and leaves, with up-regulation under Cd stress induction. Importantly, overexpressing SpSIZ1 resulted in lower Cd translocation factors (Tfs) but maintained relatively constant Cd levels in roots under Cd stress, leading to enhanced Cd stress resistance in plants. Protein interaction analysis revealed that SpSIZ1 interacts with SpABI5, and the expression of genes responsive to abscisic acid (ABA) through SpABI5-dependent signaling was significantly up-regulated in SpSIZ1-overexpressing plants with Cd stress treatment. Collectively, our results illustrate that SpSIZ1 interacts with SpABI5, enhancing the expression of ABA downstream stress-related genes through SpABI5, thereby increasing Cd tolerance in plants.

Rotation of Celosia argentea and Sedum plumbizincicola promotes Cd phytoextraction efficiency.

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.

Impacts of simulated atmospheric cadmium deposition on the physiological response and cadmium accumulation of Sedum plumbizincicola.

Atmospheric cadmium (Cd) deposition contributes to the accumulation of Cd in the soil-plant system. Sedum plumbizincicola is a Cd and Zn hyperaccumulator commonly used for the phytoremediation of Cd-contaminated soil. However, studies on the effects of atmospheric Cd deposition on the accumulation of Cd and physiological response in S. plumbizincicola are still limited. A Cd solution spraying pot experiment was conducted with S. plumbizincicola at three atmospheric Cd deposition concentrations (4, 8, and 12 mg/L). Each Cd concentration levels was divided into two groups, non-mulching (foliar-root uptake) and mulching (foliar uptake). The soil type used in the experiment was reddish clayey soil collected from a farmland. The results showed that compared with the non-mulching control, the fresh weight of S. plumbizincicola in non-mulching with high atmospheric Cd deposition (12 mg/L) increased by 11.35%. Compared with those in the control group, the malondialdehyde (MDA) content in the non-mulching and mulching S. plumbizincicola groups increased by 0.88-11.06 nmol/L and 0.96-1.32 nmol/L, respectively. Compared with those in the non-Cd-treated control group, the shoot Cd content in the mulching group significantly increased by 11.09-180.51 mg/kg. Under high Cd depositions, the Cd in S. plumbizincicola mainly originated from the air and was stored in the shoots (39.7-158.5%). These findings highlight that the physiological response and Cd accumulation of S. plumbizincicola were mainly affected by high Cd deposition and suggest that atmospheric Cd could directly be absorbed by S. plumbizincicola. The effect of atmospheric deposition on S. plumbizincicola cannot be ignored.

Does phytoextraction with Sedum plumbizincicola increase cadmium leaching from polluted agricultural soil?

Sedum plumbizincicola is a cadmium (Cd) and zinc hyperaccumulator that can activate Cd by rhizosphere acidification. However, there is little understanding of the Cd leaching risk from polluted soil during phytoextraction process. Here, pot and column experiments were conducted to monitor soil Cd leaching characteristics under different rainfall simulation conditions during S. plumbizincicola phytoextraction. Soil Cd leaching increased significantly with increasing simulated rainfall intensity. Compared with normal rainfall (NR), weak rainfall (WR) resulted in a 34.3% decrease in Cd uptake by S. plumbizincicola and also led to a 68.7% decline in Cd leaching. In contrast, Cd leaching under heavy rainfall (HR) was 2.12 times that of NR in the presence of S. plumbizincicola. After two successive growing periods, phytoextraction resulted in a 53.5-66.4% decline in the amount of soil Cd leached compared with controls in which S. plumbizincicola was absent. Even compared with maize cropping as a control, S. plumbizincicola did not instigate a significant increase in Cd leaching. The contribution of Cd leaching loss to the decline in soil total Cd concentration was negligible after phytoextraction in the pot experiment. Overall, the results contribute to our understanding of soil Cd leaching risk by phytoextraction with S. plumbizincicola.

Repeated phytoextraction by hyperaccumulator Sedum plumbizincicola is an important remediation technology to remove Cd from contaminated soils. At the same time, Sedum plumbizincicola can also activate soil Cd by rhizosphere acidification. However, studies on the leaching risk of soil activated Cd during the phytoextraction process are very few. This study looked at the effects of Sedum plumbizincicola growth on soil Cd leaching with the changes in rainfall simulation and plant type. Results showed that repeated phytoextraction with Sedum plumbizincicola did not increase Cd leaching from contaminated soil.

Cadmium levels and soil pH drive structure and function differentiation of endophytic bacterial communities in Sedum plumbizincicola: A field study.

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.

Exploring Transcriptional Regulation of Hyperaccumulation in Sedum plumbizincicola through Integrated Transcriptome Analysis and CRISPR/Cas9 Technology.

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.

A novel gene SpCTP3 from the hyperaccumulator Sedum plumbizincicola redistributes cadmium and increases its accumulation in transgenic Populus × canescens.

A cadmium (Cd) tolerance protein (SpCTP3) involved in the Sedum plumbizincicola response to Cd stress was identified. However, the mechanism underlying the Cd detoxification and accumulation mediated by SpCTP3 in plants remains unclear. We compared wild-type (WT) and SpCTP3-overexpressing transgenic poplars in terms of Cd accumulation, physiological indices, and the expression profiles of transporter genes following with 100 µmol/L CdCl2. Compared with the WT, significantly more Cd accumulated in the above-ground and below-ground parts of the SpCTP3-overexpressing lines after 100 µmol/L CdCl2 treatment. The Cd flow rate was significantly higher in the transgenic roots than in the WT roots. The overexpression of SpCTP3 resulted in the subcellular redistribution of Cd, with decreased and increased Cd proportions in the cell wall and the soluble fraction, respectively, in the roots and leaves. Additionally, the accumulation of Cd increased the reactive oxygen species (ROS) content. The activities of three antioxidant enzymes (peroxidase, catalase, and superoxide dismutase) increased significantly in response to Cd stress. The observed increase in the titratable acid content in the cytoplasm might lead to the enhanced chelation of Cd. The genes encoding several transporters related to Cd2+ transport and detoxification were expressed at higher levels in the transgenic poplars than in the WT plants. Our results suggest that overexpressing SpCTP3 in transgenic poplar plants promotes Cd accumulation, modulates Cd distribution and ROS homeostasis, and decreases Cd toxicity via organic acids. In conclusion, genetically modifying plants to overexpress SpCTP3 may be a viable strategy for improving the phytoremediation of Cd-polluted soil.

Overexpression of Sedum SpHMA2, SpHMA3 and SpNramp6 in Brassica napus increases multiple heavy metals accumulation for phytoextraction.

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.

The Mitogenome of Sedum plumbizincicola (Crassulaceae): Insights into RNA Editing, Lateral Gene Transfer, and Phylogenetic Implications.

As the largest family within the order Saxifragales, Crassulaceae contains about 34 genera with 1400 species. Mitochondria play a critical role in cellular energy production. Since the first land plant mitogenome was reported in Arabidopsis, more than 400 mitogenomic sequences have been deposited in a public database. However, no entire mitogenome data have been available for species of Crassulaceae to date. To better understand the evolutionary history of the organelles of Crassulaceae, we sequenced and performed comprehensive analyses on the mitogenome of Sedum plumbizincicola. The master mitogenomic circle is 212,159 bp in length, including 31 protein-coding genes (PCGs), 14 tRNA genes, and 3 rRNA genes. We further identified totally 508 RNA editing sites in PCGs, and demonstrated that the second codon positions of mitochondrial genes are most prone to RNA editing events. Notably, by neutrality plot analyses, we observed that the mitochondrial RNA editing events have large effects on the driving forces of plant evolution. Additionally, 4 MTPTs and 686 NUMTs were detected in the mitochondrial and nuclear genomes of S. plumbizincicola, respectively. Additionally, we conducted further analyses on gene transfer, secondary structures of mitochondrial RNAs, and phylogenetic implications. Therefore, the findings presented here will be helpful for future investigations on plant mitogenomes.

Effects of four endophytic bacteria on cadmium speciation and remediation efficiency of Sedum plumbizincicola in farmland soil.

Cadmium (Cd) pollution in farmland soils severely affects agricultural production safety, thereby threatening human health. Sedum plumbizincicola is a Cd and Zn hyperaccumulator commonly used for the phytoremediation of Cd-contaminated soil. This study was aimed to improve the remediation effect of S. plumbizincicola on Cd-contaminated farmland soil and provide a theoretical basis for the enhancement of endophytic bacteria in the repair of Cd-contaminated soil with S. plumbizincicola. Four kinds of endophytic bacteria, namely Buttiauxella, Pedobacter, Aeromonas eucrenophila, and Ralstonia pickettii, were used, and soil culture experiments and pot experiments were conducted to explore the effects of endophytic bacteria on soil Cd speciation and phytoremediation efficiency of Cd-contaminated farmland soils. Under the experimental conditions, after inoculation with endophytic bacteria, the soil pH was effectively reduced, content of weak acid-extracted Cd and oxidizable Cd increased, and content of reducible Cd and residual Cd decreased. Soil Cd activity was increased, and the availability coefficient of soil Cd increased by 1.15 to 6.41 units compared with that of the control (CK2). Compared with CK2, the biomass of S. plumbizincicola significantly increased by 23.23-55.12%; Cd content in shoots and roots of S. plumbizincicola increased by 29.63-46.01% and 11.42-84.47%, respectively; and bioconcentration factor was 2.13 to 2.72 times that of CK2. The Cd removal rate of S. plumbizincicola monocropping was 48.25%. When S. plumbizincicola was planted with inoculating endophytic bacteria, the Cd removal rate in the soil reached 61.18-71.49%, which was significantly higher than that of CK2 (p < 0.05). The treatment with endophytic bacteria activated soil Cd, promoted the growth of S. plumbizincicola, increased its Cd content, and enhanced the phytoremediation of Cd-contaminated farmland soil. Therefore, endophytic bacteria can be used to improve the remediation efficiency of S. plumbizincicola in Cd-contaminated farmland soils.

Identification and Analysis of bZIP Family Genes in Sedum plumbizincicola and Their Potential Roles in Response to Cadmium Stress.

Sedum plumbizincicola (Crassulaceae), a cadmium (Cd)/zinc (Zn)/lead (Pb) hyperaccumulator native to Southeast China, is potentially useful for the phytoremediation of heavy metal-contaminated soil. Basic leucine zipper (bZIP) transcription factors play vital roles in plant growth, development, and abiotic stress responses. However, there has been minimal research on the effects of Cd stress on the bZIP gene family in S. plumbizincicola. In this study, 92 SpbZIP genes were identified in the S. plumbizincicola genome and then classified into 12 subgroups according to their similarity to bZIP genes in Arabidopsis. Gene structure and conserved motif analyses showed that SpbZIP genes within the same subgroup shared similar intron-exon structures and motif compositions. In total, eight pairs of segmentally duplicated SpbZIP genes were identified, but there were no tandemly duplicated SpbZIP genes. Additionally, the duplicated SpbZIP genes were mainly under purifying selection pressure. Hormone-responsive, abiotic and biotic stress-responsive, and plant development-related cis-acting elements were detected in the SpbZIP promoter sequences. Expression profiles derived from RNA-seq and quantitative real-time PCR analyses indicated that the expression levels of most SpbZIP genes were upregulated under Cd stress conditions. Furthermore, a gene co-expression network analysis revealed that most edge genes regulated by hub genes were related to metal transport, responses to stimuli, and transcriptional regulation. Because its expression was significantly upregulated by Cd stress, the hub gene SpbZIP60 was selected for a functional characterization to elucidate its role in the root response to Cd stress. In a transient gene expression analysis involving Nicotiana benthamiana leaves, SpbZIP60 was localized in the nucleus. The overexpression of SpbZIP60 enhanced the Cd tolerance of transgenic Arabidopsis plants by inhibiting ROS accumulation, protecting the photosynthetic apparatus, and decreasing the Cd content. These findings may provide insights into the potential roles of the bZIP family genes during the S. plumbizincicola response to Cd stress.

Effects of a soil collembolan on the growth and metal uptake of a hyperaccumulator: Modification of root morphology and the expression of plant defense genes.

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.

Genome-Wide Characterization of Sedum plumbizincicola HMA Gene Family Provides Functional Implications in Cadmium Response.

Heavy-metal ATPase (HMA), an ancient family of transition metal pumps, plays important roles in the transmembrane transport of transition metals such as Cu, Zn, Cd, and Co. Although characterization of HMAs has been conducted in several plants, scarcely knowledge was revealed in Sedum plumbizincicola, a type of cadmium (Cd) hyperaccumulator found in Zhejiang, China. In this study, we first carried out research on genome-wide analysis of the HMA gene family in S. plumbizincicola and finally identified 8 SpHMA genes and divided them into two subfamilies according to sequence alignment and phylogenetic analysis. In addition, a structural analysis showed that SpHMAs were relatively conserved during evolution. All of the SpHMAs contained the HMA domain and the highly conserved motifs, such as DKTGT, GDGxNDxP, PxxK S/TGE, HP, and CPx/SPC. A promoter analysis showed that the majority of the SpHMA genes had cis-acting elements related to the abiotic stress response. The expression profiles showed that most SpHMAs exhibited tissue expression specificity and their expression can be regulated by different heavy metal stress. The members of Zn/Co/Cd/Pb subgroup (SpHMA1-3) were verified to be upregulated in various tissues when exposed to CdCl2. Here we also found that the expression of SpHMA7, which belonged to the Cu/Ag subgroup, had an upregulated trend in Cd stress. Overexpression of SpHMA7 in transgenic yeast indicated an improved sensitivity to Cd. These results provide insights into the evolutionary processes and potential functions of the HMA gene family in S. plumbizincicola, laying a theoretical basis for further studies on figuring out their roles in regulating plant responses to biotic/abiotic stresses.

Integrative analysis of transcriptome and proteome provides insights into adaptation to cadmium stress in Sedum plumbizincicola.

Sedum plumbizincicola, a cadmium (Cd) hyperaccumulating herbaceous plant, can accumulate large amounts of Cd in the above-ground tissues without being poisoned. However, the molecular mechanisms regulating the processes are not fully understood. In this study, Transcriptional and proteomic analyses were integrated to investigate the response of S. plumbizincicola plants to Cd stress and to identify key pathways that are potentially responsible for Cd tolerance and accumulation. A total of 630 DAPs (differentially abundant proteins, using fold change >1.5 and adjusted p-value <0.05) were identified from Tandem Mass Tag (TMT)- based quantitative proteomic profiling, which were enriched in processes including phenylpropanoid biosynthesis, protein processing in endoplasmic reticulum, and biosynthesis of secondary metabolites. Combined with the previous transcriptomic study, 209 genes and their corresponding proteins showed the identical expression pattern. The identified genes/proteins revealed the potential roles of several metabolism pathways, including phenylpropanoid biosynthesis, oxidative phosphorylation, phagosome, and glutathione metabolism, in mediating Cd tolerance and accumulation. Lignin staining and Cd accumulation assay of the transgenic lines over-expressing a selected Cd up-regulated gene SpFAOMT (Flavonoid 3',5'-methyltransferase) showed its functions in adapting to Cd stress, and provided insight into its role in lignin biosynthesis and Cd accumulation in S. plumbizincicola during Cd stress.

A field study reveals links between hyperaccumulating Sedum plants-associated bacterial communities and Cd/Zn uptake and translocation.

Hyperaccumulating ecotypes of Sedum plants are promising Cd/Zn phytoextractors, with potential for leveraging its rhizospheric or endophytic microbiomes to improve phytoremediation efficiency. However, research of bacteria associated with Sedum at field scale is still lacking. Here, we presented a detailed investigation of the bacterial microbiome of hyperaccumulating Sedum ecotypes (S. alfredii and S. plumbizincicola) and a non-hyperaccumulating S. alfredii ecotype, which grow at different soil environments. Moreover, we evaluated the heavy metal uptake and translocation potential of Sedum plants at different locations. The results showed that both HE ecotypes, contrary to the NHE, were efficient for phytoremediation in mine areas and farmlands. For NHE plants, rhizosphere co-occurrence networks were more complex than the networks of other compartments, while networks of HE plants were more complex in bulk soil and roots. The proportion of positive correlations within co-occurrence networks was higher for the HE plants, suggesting a greater potential for mutualistic interactions. Plant compartment and location predominantly shaped the microbiome assembly, and Proteobacteria, Actinobacteria and Acidobacteria dominated the bacterial communities of Sedum plants. Keystone taxa related to Zn hyperaccumulation are similar to those related to Cd hyperaccumulation, and nine bacterial genera had significantly positive correlation with Cd/Zn hyperaccumulation. Taxa, linked to phytoremediation in both mine and farmland (i.e. Actinospica and Streptomyces from Actinobacteria), could be targets for further investigation of their ability to promote metal phytoremediation of Sedum species.

Zinc uptake and replenishment mechanisms during repeated phytoextraction using Sedum plumbizincicola revealed by stable isotope fractionation.

Improving phytoremediation techniques requires a thorough understanding of the mechanisms of plant uptake and the replenishment of the bioavailable pool of the target element, and this may be effectively explored using stable isotope methods. A repeated phytoextraction experiment over five successive crops of cadmium (Cd) and zinc (Zn) hyperaccumulator Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu (Crassulaceae) was conducted using four agricultural soils differing in soil pH and clay content. The isotopic composition of total Zn and NH4OAc-extractable Zn in soils before phytoextraction and after the fifth crop were determined, together with Zn in shoot samples in the first crop. S. plumbizincicola preferentially took up light Zn isotopes from the NH4OAc-extractable pool (Δ66Znshoot-extract = -0.42 to -0.16‰), indicating the predominance of Zn low-affinity transport. However, after long-term phytoextraction NH4OAc-extractable Zn became isotopically lighter than prior to phytoextraction in three of the soils (Δ66Znextract: P5-P0 = -0.39 to -0.10‰). This was resulted from the equilibrium replenishment of Zn bound to iron (Fe) and manganese (Mn) oxides based on Zn isotopic and chemical speciation analysis. Zinc showed opposite fractionation patterns to Cd in the same plant-soil system with heavy Cd isotope enrichment in S. plumbizincicola (Δ114/110Cdshoot-extract = 0.02-0.17‰) and in the NH4OAc-extractable pool after repeated phytoextraction (Δ114/110Cdextract: P5-P0 = 0.07-0.18‰). This indicates different mechanisms of membrane transport (high-affinity transport of Cd) and supplementation of the bioavailable pool in soil (Cd supplied mainly through complexation with root-derived organic ligands) of the two metals. The combination of chemical speciation and stable Zn isotope ratios in the plant and the bioavailable soil pool reveal that the Zn pool related to Fe and Mn oxides became increasingly bioavailable with increasing crop generations. Capsule: Stable isotope analysis indicates that soil Fe- and Mn-oxide bound Zn replenishment boosted Zn uptake by the hyperaccumulator Sedum plumbizincicola during long-term remediation.

Dispose waste liquor of fresh biomass of a hyperaccumulator Sedum plumbizincicola in phytoextraction process.

Sedum plumbizincicola has been widely employed to remove cadmium (Cd) and zinc (Zn) from contaminated soils and harvested biomass is used to recover valuable metals. While chopping and compacting are efficient methods to rapidly reduce the volume and moisture of fresh biomass, the resulting waste liquor containing metals needs treatment. Two types of contaminated soils were cropped with S. plumbizincicola and top-dressed with this liquor to study metals migration in soil profile and their uptake by plants. There were three treatments: planting and adding liquor (PL), planting without liquor (P) and adding liquor without planting (L). The results showed that Cd and Zn from liquor were mainly retained at top soil 0-10 cm under L treatment. Compared with L treatment, soil Cd and Zn under PL treatment decreased significantly in soil profile due to the extraction of S. plumbizincicola. Moreover, the amount of Cd and Zn extracted by plants was greater than that applied in soils. The metal removal rate by S. plumbizincicola in acid clay loam soil was higher than that in neutral sandy soil. To sum up, metal retaining in soil and uptake by S. plumbizincicola can be used to treat waste liquor from its fresh biomass. Novelty StatementRapid and efficient treatment of harvested fresh biomass is still a challenge although phytoextraction using hyperaccumulator Sedum plumbizincicola has been widely employed. Chopping and compacting fresh biomass are efficient methods for rapid dehydration, however, a large amount of waste liquor that contains of Cd and Zn is produced and needs treatment. In the present study, a simple and low-cost method was tested to dispose the liquor, i. e. irrigating it onto the surface of contaminated soils where grown S. plumbizincicola. It was found that Cd and Zn applied in soils from liquor were mainly retained at top 0-10 cm soil depth where S. plumbizincicola root system was widespread, and the amount of Cd and Zn extracted by plants was greater than that applied in soils. Therefore, it is technically feasible to dispose the waste liquor dewatering from fresh biomass of S. plumbizincicola in its phytoextraction process. This study is helpful for the rapid, efficient and low-cost treatment of harvested fresh biomass in the large-scale application of phytoremediation.

Quantitative proteome analysis reveals changes of membrane transport proteins in Sedum plumbizincicola under cadmium stress.

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.

Phytoremediation potential of wheat intercropped with different densities of Sedum plumbizincicola in soil contaminated with cadmium and zinc.

Intercropping technology is applied widely in crop cultivation to help remediate soil polluted with heavy metals. To investigate the feasibility and potential of intercropping hyperaccumulator plants with crops in cadmium (Cd)- and zinc (Zn)-contaminated soil, a pot experiment was conducted to examine plant growth and the contents of Cd and Zn in the soil following intercropping of wheat and Sedum plumbizincicola. Five treatments were examined: control (wheat monoculture: 36 seedlings per pot), and intercropping of wheat with different planting densities of S. plumbizincicola (3, 6, 9 and 15 seedlings per pot, respectively). Results showed a decrease in soil pH, and in soil and wheat contents of Cd and Zn with increasing planting density of S. plumbizincicola, while the removal rate of Cd and Zn increased. Meanwhile, excessive planting (15 seedlings per pot) inhibited wheat growth by 27.34% compared with the control, and overall, the optimal planting density was 9 seedlings per pot, resulting in effective remediation with only a moderate effect on wheat growth. These findings highlight the value of intercropping S. plumbizincicola with wheat as a means of improving remediation of soil contaminated with heavy metals (Cd and Zn).