SpCTP3 from the hyperaccumulator Sedum plumbizincicola positively regulates cadmium tolerance by interacting with SpMDH1.

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 Cd2+ binding and thermodynamic analyses, revealed the CXXC motif in SpCTP3 functions is a Cd2+ 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.

Physiology and transcriptomic analysis revealed the mechanism of silicon promoting cadmium accumulation in Sedum alfredii Hance.

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.

Salicylic Acid's impact on Sedum alfredii growth and cadmium tolerance: Comparative physiological, transcriptomic, and metabolomic study.

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.

Intraspecific and Intrageneric Genomic Variation across Three Sedum Species (Crassulaceae): A Plastomic Perspective.

Sedum is the largest succulent genus in Crassulaceae. Because of predominant maternal inheritance, little recombination, and slow evolution, plastomes can serve as powerful super barcodes for inter- or intra-species phylogenetic analyses. While previous research has focused on plastomes between Sedum species, intra-species studies are scarce. Here, we sequenced plastomes from three Sedum species (Sedum alfredii, Sedum plumbizincicola, and Sedum japonicum) to understand their evolutionary relationships and plastome structural evolution. Our analyses revealed minimal size and GC content variation across species. However, gene distribution at IR boundaries, repeat structures, and codon usage patterns showed diversity at both inter-specific and intra-specific levels. Notably, an rps19 gene expansion and a bias toward A/T-ending codons were observed. Codon aversion motifs also varied, potentially serving as markers for future studies. Phylogenetic analyses confirmed the non-monophyly of Sedum and divided the Acre clade into two groups. Individuals from the same species clustered together, with strong support for the relationships between S. alfredii, S. tricarpum, and S. plumbizincicola. Additionally, S. japonicum clearly affiliates with the Acre clade. This study provides valuable insights into both intra-specific and intra-generic plastome variation in Sedum, as well as overall plastome evolution within the genus.

Ectopic expression of SaCTP3 from the hyperaccumulator Sedum alfredii in sorghum increases Cd accumulation for phytoextraction.

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.

Cadmium inhibits powdery mildew colonization and reconstructs microbial community in leaves of the hyperaccumulator plant Sedum alfredii.

Understanding the influence of the heavy metal cadmium (Cd) on the phyllosphere microbiome of hyperaccumulator plants is crucial for enhancing phytoremediation. The characteristics of the phyllosphere of Sedum alfredii Hance, a hyperaccumulator plant, were investigated using 16S rRNA and internal transcribed spacer amplicon sequencing of powdery mildew-infected leaves treated or untreated with Cd. The results showed that the colonization of powdery mildew caused severe chlorosis and necrosis in S. alfredii leaves, and the relative abundance of Leotiomycetes in infected leaves increased dramatically and significantly decreased phyllosphere microbiome diversity. However, S. alfredii preferentially accumulated higher concentrations of Cd in the leaves of infected plants than in uninfected plants by powdery mildew, which in turn significantly inhibited powdery mildew colonization in leaves; the relative abundance of the fungal class Leotiomycetes in infected leaves decreased, and alpha and beta diversities of the phyllosphere microbiome significantly increased with Cd treatment in the infected plants. In addition, the inter-kingdom networks in the microbiota of the infected leaves treated with Cd presented many nodes and edges, and the highest inter-kingdom modularity compared to the untreated infected leaves, indicating a highly connected microbial community. These results suggest that Cd significantly inhibits powdery mildew colonization by altering the composition of the phyllosphere microbiome in S. alfredii leaves, paving the way for efficient heavy metal phytoremediation and providing a new perspective on defense strategies against heavy metals.

Functional characterization of a DNA-damage repair/tolerance 100 (DRT100) gene in Sedum alfredii Hance for genome stability maintenance and Cd hypertolerance.

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.

SaMT3 in Sedum alfredii drives Cd detoxification by chelation and ROS-scavenging via Cys residues.

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.

Transcriptome analysis reveals candidate genes involved in multiple heavy metal tolerance in hyperaccumulator Sedum alfredii.

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.

The involvement of nitric oxide and ethylene on the formation of endodermal barriers in response to Cd in hyperaccumulator Sedum alfredii.

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.

Genome-wide characterization of the hyperaccumulator Sedum alfredii F-box family under cadmium stress.

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

Auxin-Induced SaARF4 Downregulates SaACO4 to Inhibit Lateral Root Formation in Sedum alfredii Hance.

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.

Identification and characterization of SaeIF1 from the eukaryotic translation factor SUI1 family in cadmium hyperaccumulator Sedum alfredii.

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.

Ethylene-mediated apoplastic barriers development involved in cadmium accumulation in root of hyperaccumulator Sedum alfredii.

Ethylene is an important phytohormone for plant adaptation to heavy metal stress. However, the effects of ethylene on radial apoplastic transport of Cd remain elusive. This study investigated the role of ethylene on apoplastic barriers development and consequences for Cd uptake in Sedum alfredii. In response to Cd, endogenous ethylene production in hyperaccumulating ecotype (HE) roots was decreased due to the down-regulated expressions of ethylene biosynthesis genes, while the opposite result was observed in non-hyperaccumulating ecotype (NHE). Interestingly, the ethylene emission in HE was always higher than that in NHE, regardless of Cd concentrations. Results of exogenous application of ethylene biosynthesis precursor/inhibitor indicate that ethylene with high level would delay the formation of apoplastic barriers in HE through restraining phenylalanine ammonia lyase activity and gene expressions related to lignin/suberin biosynthesis. Simultaneously, correlation analyses suggest that Cd-induced apoplastic barriers formation may be also regulated by ethylene signaling. By using an apoplastic bypass tracer and scanning ion-selected electrode, we observed that the delayed deposition of apoplastic barriers significantly promoted Cd influx in roots. Taken together, high endogenous ethylene in HE postponed the formation of apoplastic barriers and thus promoted the Cd accumulation in the apoplast of roots.

Identification and functional characterization of ABCC transporters for Cd tolerance and accumulation in Sedum alfredii Hance.

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

A Single Amino Acid Change in Nramp6 from Sedum Alfredii Hance Affects Cadmium Accumulation.

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 SaNramp6hL157P 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.

[Isolation and characterization of cadmium tolerant gene SpMT2 in the hyperaccumulator Sedum plumbizincicola].

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

Chromosome doubling of Sedum alfredii Hance: A novel approach for improving phytoremediation efficiency.

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.

Time of day and network reprogramming during drought induced CAM photosynthesis in Sedum album.

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.

Transcriptome sequencing and functional analysis of Sedum lineare Thunb. upon salt stress.

Soil salinization is one major constraint to plant geographical distribution, yield, and quality, and as an ideal plant for the "greening" of flat-roofed buildings, Sedum lineare Thunb. has strong tolerance against a variety of environmental adversities including salinity with the underlying mechanism still remaining unknown. In this study, we performed de novo transcriptome sequencing on leaf and root samples of NaCl-treated S. lineare Thunb. and identified 584 differentially expressed genes (DEGs), which were further annotated by gene function classification and pathway assignments using the public data repositories. In addition to the increased gene expression level verified by qRT-PCR, the elevated activities of the corresponding enzymes were also demonstrated for peroxidase (POD), glutathione peroxidases (GPX), and cysteine synthase (CSase) in the NaCl-treated roots. Furthermore, two highly inducible genes without known functions related to salt tolerance were selected to be overexpressed and tested for their effects on salt tolerance in the model plant, Arabidopsis thaliana. Upon 150 mM NaCl treatment, 35S:SlCXE but not 35S:SlCYP72A transgenic Arabidopsis seedlings exhibited improved salt resistance as shown by the increased seed germination rates and longer primary roots of transgenic seedlings when compared to wild-type plants. Taken together, this work laid a foundation for a better understanding of the salt adaptation mechanism of S. lineare Thunb. and genes identified could serve as useful resources for the development of more salt-tolerant varieties of other species through genetic engineering.