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.

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.

Exogenous abscisic acid (ABA) promotes cadmium (Cd) accumulation in Sedum alfredii Hance by regulating the expression of Cd stress response genes.

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.

SpHMA1 is a chloroplast cadmium exporter protecting photochemical reactions in the Cd hyperaccumulator Sedum plumbizincicola.

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.

Effects of CO2 application and endophytic bacterial inoculation on morphological properties, photosynthetic characteristics and cadmium uptake of two ecotypes of Sedum alfredii Hance.

Plant uptake of cadmium (Cd) is affected by soil and environmental conditions. In this study, hydroponic experiments were conducted to investigate the effects of elevated CO2 coupled with inoculated endophytic bacteria M002 on morphological properties, gas exchange, photosynthetic pigments, chlorophyll fluorescence, and Cd uptake of S. alfredii. The results showed that bio-fortification processes (elevated CO2 and/or inoculated with endophytic bacteria) significantly (p < 0.05) promoted growth patterns, improved photosynthetic characteristics and increased Cd tolerance of both ecotypes of S. alfredii, as compared to normal conditions. Net photosynthetic rate (Pn) in intact leaves of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) were increased by 73.93 and 32.90%, respectively at the low Cd (2 µM), 84.41 and 57.65%, respectively at the high Cd level (10 µM). Superposition treatment increased Cd concentration in shoots and roots of HE, by 50.87 and 82.12%, respectively at the low Cd and 46.75 and 88.92%, respectively at the high Cd level. Besides, superposition treatment declined Cd transfer factor of NHE, by 0.85% at non-Cd rate, 17.22% at the low Cd and 22.26% at the high Cd level. These results indicate that elevated CO2 coupled with endophytic bacterial inoculation may effectively improve phytoremediation efficiency of Cd-contaminated soils by hyperaccumulator, and alleviate Cd toxicity to non-hyperaccumulator ecotype of Sedum alfredii.

Identification, Expression Analysis of the Hsf Family, and Characterization of Class A4 in Sedum Alfredii Hance under Cadmium Stress.

Sedum alfredii Hance, a cadmium (Cd)/zinc (Zn)/lead (Pb) co-hyperaccumulating species, is a promising phytoremediation candidate because it accumulates substantial amounts of heavy metal ions without showing any obvious signs of poisoning. The heat shock transcription factor (Hsf) family plays crucial roles in plant growth, development, and stress responses. Although the roles of some Hsfs in abiotic stress have been well studied in model plants, the Hsf family has not been systematically investigated in heavy metal hyperaccumulators. Here, we comprehensively analyzed the Hsf gene family in S. alfredii based on a transcriptome under Cd stress. There were 22 Hsf members that were identified and phylogenetically clustered into three classes, namely, SaHsfA, SaHsfB, and SaHsfC. All of the three classes shared similar motifs. The expression profiles of the 22 Hsf members showed significant differences: 18 SaHsfs were responsive to Cd stress, as were multiple SaHsp genes, including SaHsp18.1, SaHsp22, SaHsp26.5, SaHsp70, SaHsp90, and SaHsp101. Two class A4 members, SaHsfA4a and SaHsfA4c, exhibited transcriptional activation activities. Overexpression of SaHsfA4a and SaHsfA4c in transgenic yeast indicated an improved tolerance to Cd stress and Cd accumulation. Our results suggest SaHsfs play important regulatory roles in heavy metal stress responses, and provide a reference for further studies on the mechanism of heavy metal stress regulation by SaHsfs.

[Impacts of Vegetation on Hydrological Performances of Green Roofs Under Different Rainfall Conditions].

Vegetation is an important component of green roofs and may affect their hydrological performance through the processes of rainwater interception and evapotranspiration. Based on the rainfall-runoff observations of green roofs with four types of vegetation covers (Portulaca grandiflora, Sedum lineare, Festuca elata, and bare substrate) located in Beijing during 26 rainfall events from April to October 2017, the impacts of vegetation cover on the hydrological performance of green roofs were investigated using runoff and peak discharge reduction rates and time-delay of runoff generation and peak discharge as indices. For the 12 green roofs, there was a significantly negative correlation (P<0.01) between runoff reduction rate and rainfall event volume. For low rainfall (<10 mm), the runoff reduction rates of all the green roofs were equal or close to 100%. When the rainfall volume increased to about 30 mm, the runoff reduction rates dropped to below 70%. For the heaviest rainfall event during the observation period (81.4 mm), the runoff reduction rates of all the green roofs were less than 55%. The impacts of vegetation on the hydrological performance of green roofs changed with rainfall conditions. The differences between runoff reduction rates of green roofs with different types of vegetation cover were largest for the heavy rainfall events. For the moderate rainstorm events, the differences were slightly lower. For light rainfall events, however, no significant differences were observed among the runoff reduction rates of green roofs with different types of vegetation cover, as little runoff was generated. Vegetation cover could enhance the hydrological performance of green roofs, as the runoff and peak discharge reduction rates and time-delay of runoff generation and peak discharge of green roofs covered with vegetation were all better than those of the bare substrate for all the groups of rainfall events except the light rainfall. Vegetation-covered green roofs with P. grandiflora performed the best, as the average height and aboveground biomass per unit area of P. grandiflora were the largest.

Phylogeography of a tough rock survivor in European dry grasslands.

Phylogeographic analyses of plants in Europe have revealed common glacial refugia and migration routes for several trees and herbs with arctic-alpine distributions. The postglacial histories of dry grassland species in central Europe have rarely been analyzed, even though the extremely species-rich habitat is threatened. Sedum album (Crassulaceae) is a common inhabitant of rocky sites in central European dry grasslands. We inferred the phylogeographic history of S. album over its distribution range in Europe. Genetic diversity within and differentiation between 34 S. album populations was examined using AFLP markers. Population isolation was indicated based on the rarity of the fragments and by isolation-by-distance effects. We sequenced the trnL-trnF region in 32 populations and used chloroplast microsatellites to analyze chloroplast haplotype distributions. Two distinct S. album lineages were detected. One lineage was comprised of populations from eastern and central parts of central Europe, and the Apennine Peninsula. A second lineage was comprised of populations from the Iberian Peninsula and western and northern parts of central Europe. Glacial refugia were identified based on the accumulation of ancient chloroplast haplotypes, high diversity of AFLP fragments within populations, and high levels of rare fragments in Liguria, Serbia, the Apennine and Iberian peninsulas. Cryptic refugia were detected in the Czech Republic and Slovakia. Isolation by distance was present all over the distribution range, and it was separately detected in southwestern and central Europe. In western Europe, where a contact zone between the two lineages can be expected, no isolation by distance was detected. Our results suggest migration routes of S. album northeastward from glacial refugia in southern Iberia, northward from the Apennine Peninsula, and northward and westward from the southeastern parts of central Europe. Therefore, central European grasslands were recently colonized by northern cryptic populations and source populations originating in the east and the Apennine Peninsula.

Unique parallel radiations of high-mountainous species of the genus Sedum (Crassulaceae) on the continental island of Taiwan.

We explored the temporal and spatial diversification of the plant genus Sedum L. (Crassulaceae) in Taiwan based on molecular analysis of nrITS and cpDNA sequences from East Asian Sedum members. Our phylogenetic and ancestral area reconstruction analysis showed that Taiwanese Sedum comprised two lineages that independently migrated from Japan and Eastern China. Furthermore, the genetic distances among species in these two clades were smaller than those of other East Asian Sedum clades, and the Taiwanese members of each clade occupy extremely varied habitats with similar niches in high-mountain regions. These data indicate that species diversification occurred in parallel in the two Taiwanese Sedum lineages, and that these parallel radiations could have occurred within the small continental island of Taiwan. Moreover, the estimated time of divergence for Taiwanese Sedum indicates that the two radiations might have been correlated to the formation of mountains in Taiwan during the early Pleistocene. We suggest that these parallel radiations may be attributable to the geographical dynamics of Taiwan and specific biological features of Sedum that allow them to adapt to new ecological niches.

Enhanced metal tolerance correlates with heterotypic variation in SpMTL, a metallothionein-like protein from the hyperaccumulator Sedum plumbizincicola.

Mechanistic insight into metal hyperaccumulation is largely restricted to Brassicaceae plants; therefore, it is of great importance to obtain corresponding knowledge from system outside the Brassicaceae. Here, we constructed and screened a cDNA library of the Cd/Zn hyperaccumulator Sedum plumbizincicola and identified a novel metallothionein-like protein encoding gene SpMTL. SpMTL showed functional similarity to other known MT proteins and also to its orthologues from non-hyperaccumulators. However, three additional cysteine residues were observed in SpMTL and appeared to be hyperaccumulator specific. Removal of these three residues significantly decreased its ability to tolerate Cd and the stoichiometry of Cd against SpMTL (molar ratio of Cd/SpMTL) to a level comparable to those of Cd/SaMTL and Cd/SeMTL in the corresponding non-hyperaccumulating relatives. SpMTL expressed in S. plumbizincicola roots at a much higher level than those of its orthologues in the non-hyperaccumulator roots. Interestingly, a positive correlation was observed between transcript levels of SpMTL in roots and Cd accumulation in leaves. Taking these results together, we propose that elevated transcript levels and heterotypic variation in protein sequences of SpMTL might contribute to the trait of Cd hyperaccumulation and hypertolerance in S. plumbizincicola.

Characteristics of metal-tolerant plant growth-promoting yeast (Cryptococcus sp. NSE1) and its influence on Cd hyperaccumulator Sedum plumbizincicola.

Plant growth-promoting yeasts are often over looked as a mechanism to improve phytoremediation of heavy metals. In this study, Cryptococcus sp. NSE1, a Cd-tolerant yeast with plant growth capabilities, was isolated from the rhizosphere of the heavy metal hyperaccumulator Sedum plumbizincicola. The yeast exhibited strong tolerance to a range of heavy metals including Cd, Cu, and Zn on plate assays. The adsorption rate Cd, Cu, Zn by NSE1 was 26.1, 13.2, and 25.2 %, respectively. Irregular spines were formed on the surface of NSE1 when grown in MSM medium supplemented with 200 mg L(-1) Cd. NSE1 was capable of utilizing 1-aminocyclopropane-1-carboxylate (ACC) as a sole nitrogen source and was capable of solubilization of inorganic phosphate at rates of 195.2 mg L(-1). Field experiments demonstrated that NSE1 increased phytoremediation by increasing the biomass of Cd hyperaccumulator S. plumbizincicola (46 %, p < 0.05) during phytoremediation. Overall, Cd accumulation by S. plumbizincicola was increased from 19.6 to 31.1 mg m(-2) though no difference in the concentration of Cd in the shoot biomass was observed between NSE1 and control. A Cd accumulation ratio of 38.0 % for NSE1 and 17.2 % for control was observed. The HCl-extractable Cd and CaCl2-extractable Cd concentration in the soil of the NSE1 treatment were reduced by 39.2 and 29.5 %, respectively. Community-level physiology profiling, assessed using Biolog Eco plates, indicated functional changes to the rhizosphere community inoculated with NSE1 by average well color development (AWCD) and measurement of richness (diversity). Values of Shannon-Weiner index, Simpson index, and McIntosh index showed a slight but no significant increases. These results indicate that inoculation of NSE1 could increase the shoot biomass of S. plumbizincicola, enhance the Cd accumulation in S. plumbizincicola, and decrease the available heavy metal content in soils significantly without overall significant changes to the microbial community.

Enhanced expression of SaHMA3 plays critical roles in Cd hyperaccumulation and hypertolerance in Cd hyperaccumulator Sedum alfredii Hance.

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

Plant functional traits predict green roof ecosystem services.

Plants make important contributions to the services provided by engineered ecosystems such as green roofs. Ecologists use plant species traits as generic predictors of geographical distribution, interactions with other species, and ecosystem functioning, but this approach has been little used to optimize engineered ecosystems. Four plant species traits (height, individual leaf area, specific leaf area, and leaf dry matter content) were evaluated as predictors of ecosystem properties and services in a modular green roof system planted with 21 species. Six indicators of ecosystem services, incorporating thermal, hydrological, water quality, and carbon sequestration functions, were predicted by the four plant traits directly or indirectly via their effects on aggregate ecosystem properties, including canopy density and albedo. Species average height and specific leaf area were the most useful traits, predicting several services via effects on canopy density or growth rate. This study demonstrates that easily measured plant traits can be used to select species to optimize green roof performance across multiple key services.

Effect of elevated CO2 concentration on photosynthetic characteristics of hyperaccumulator Sedum alfredii under cadmium stress.

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

Repeated phytoextraction of four metal-contaminated soils using the cadmium/zinc hyperaccumulator Sedum plumbizincicola.

A cadmium/zinc hyperaccumulator extracted metals from four contaminated soils over three years in a glasshouse experiment. Changes in plant metal uptake and soil total (aqua regia-extractable) and available metals were investigated. Plant Cd concentrations in a high-Cd acid soil and plant Zn concentrations in two acid soils decreased during repeated phytoextraction and were predicted by soil available metal concentrations. However, on repeated phytoextraction, plant Cd concentrations remained constant in lightly Cd-polluted acid soils, as did plant Cd and Zn in alkaline soils, although soil available metal concentrations decreased markedly. After phytoextraction acid soils showed much higher total metal removal efficiencies, indicating possible suitability of phytoextraction for acid soils. However, DGT-testing, which takes soil metal re-supply into consideration, showed substantial removal of available metal and distinct decreases in metal supply capacity in alkaline soils after phytoextraction, suggesting that a strategy based on lowering the bioavailable contaminant might be feasible.

Elevated CO2 concentration increase the mobility of Cd and Zn in the rhizosphere of hyperaccumulator Sedum alfredii.

The effects of elevated CO2 on metal species and mobility in the rhizosphere of hyperaccumulator are not well understood. We report an experiment designed to compare the effects of elevated CO2 on Cd/Zn speciation and mobility in the rhizosphere of hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of Sedum alfredii grown under ambient (350 µl l(-1)) or elevated (800 µl l(-1)) CO2 conditions. No difference in solution pH of NHE was observed between ambient and elevated CO2 treatments. For HE, however, elevated CO2 reduced soil solution pH by 0.22 unit, as compared to ambient CO2 conditions. Elevated CO2 increased dissolved organic carbon (DOC) and organic acid levels in soil solution of both ecotypes, but the increase in HE solution was much greater than in NHE solution. After the growth of HE, the concentrations of Cd and Zn in soil solution decreased significantly regardless of CO2 level. The visual MINTEQ speciation model predicted that Cd/Zn-DOM complexes were the dominant species in soil solutions, followed by free Cd(2+) and Zn(2+) species for both ecotypes. However, Cd/Zn-DOM complexes fraction in soil solution of HE was increased by the elevated CO2 treatment (by 8.01 % for Cd and 8.47 % for Zn, respectively). Resin equilibration experiment results indicated that DOM derived from the rhizosphere of HE under elevated CO2 (HE-DOM-E) (90 % for Cd and 73 % for Zn, respectively) showed greater ability to form complexes with Cd and Zn than those under ambient CO2 (HE-DOM-A) (82 % for Cd and 61 % for Zn, respectively) in the undiluted sample. HE-DOM-E showed greater ability to extract Cd and Zn from soil than HE-DOM-A. It was concluded that elevated CO2 could increase the mobility of Cd and Zn due to the enhanced formation of DOM-metal complexes in the rhizosphere of HE S. alfredii.

Simultaneous determination by HPLC of quercetin and kaempferol in three Sedum medicinal plants harvested in different seasons.

A high-performance liquid chromatography method was established for the fast quantification of quercetin and kaempferol in three Sedum crude medicines: Sedi Herba (Sedum sarmentosum Bunge.), Sedi Linearis Herba (Sedum lineare Thunb.) and Sedi Emarginati Herba (Sedum emarginatum Migo.). The column used was a YMC-pack ODS-A (250 × 4.6 mm, 5 µm), the mobile phase was a solution of methanol-0.4% phosphoric acid (47:53) with a flow rate of 1.0 mL/min at 35°C and the detection wavelength was 360 nm. The calibration curves for quercetin and kaempferol were linear over the range of 0.01-0.62 µg for quercetin and 0.02-0.78 µg for kaempferol, and the average recoveries were 99.72% [relative standard deviation (RSD): 1.63% and 99.50% (RSD: 1.16%), respectively]. In conclusion, the method established in this paper is accurate and repeatable. It can be used for the determination of quercetin and kaempferol, controlling the quality of the three crude drugs. Furthermore, the experimental data showed that the best harvest season for the three Sedum medicinal species should be the full-bloom period between the end of April and the beginning of May.

Transcriptomic analysis of cadmium stress response in the heavy metal hyperaccumulator Sedum alfredii Hance.

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

Arbuscular mycorrhizal fungi enhance both absorption and stabilization of Cd by Alfred stonecrop (Sedum alfredii Hance) and perennial ryegrass (Lolium perenne L.) in a Cd-contaminated acidic soil.

A greenhouse pot experiment was conducted to compare the phytoextraction efficiencies of Cd by hyper-accumulating Alfred stonecrop (Sedum alfredii Hance) and fast-growing perennial ryegrass (Lolium perenne L.) from a Cd-contaminated (1.6 mg kg(-1)) acidic soil, and their responses to the inoculations of two arbuscular mycorrhizal (AM) fungal strains, Glomus caledonium 90036 (Gc) and Glomus mosseae M47V (Gm). Ryegrass and stonecrop were harvested after growing for 9 and 27 wk, respectively. Without AM fungal inoculation, the weekly Cd extraction by stonecrop (8.0 µg pot(-1)) was 4.3 times higher than that by ryegrass (1.5 µg pot(-1)). Both Gc and Gm significantly increased (P < 0.05) root mycorrhizal colonization rates, soil acid phosphatase activities, and available P concentrations, and thereby plant P absorptions (except for Gm-inoculated ryegrass), shoot biomasses, and Cd absorptions (except for Gm-inoculated stonecrop), while only Gc-inoculated stonecrop significantly accelerated (P < 0.05) the phytoextraction efficiency of Cd by 78%. In addition, both Gc and Gm significantly decreased (P < 0.05) phytoavailable Cd concentrations by 21-38% via elevating soil pH. The results suggested the potential application of hyper-accumulating Alfred stonecrop associated with AM fungi (notably Gc) for both extraction and stabilization of Cd in the in situ treatment of Cd-contaminated acidic soil.