Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri

Although Arabidopsis halleri ( = Cardaminopsis halleri) is known as a Zn hyperaccumulator, there have been no detailed studies on Zn accumulation, tolerance and cellular distribution in this species. In a hydroponic experiment, A. halleri grew healthily with Zn concentrations varying from 1 to 1000 μ M , without showing phytotoxicity or reduction in root or shoot dry weights. The concentration of Zn in the shoots increased from 300 μ g g ^{? 1} dry weight in the 1 μ M Zn treatment to 32 000 μ g g ^{? 1} in the 1000 μ M Zn treatment. Approximately 60% of the total Zn in the shoots were water‐soluble, and there was no evidence of Zn and P co‐precipitation. Both citric and malic acid concentrations in the shoots were not significantly affected by the Zn treatments, whereas in the roots there was a positive response in both organic acids to increasing Zn in solution. Cellular distribution of Zn, Ca and K in frozen hydrated leaf tissues was examined using energy‐dispersive X‐ray microanalysis. Zinc was sequestered in the base of trichomes, whereas the middle and upper parts of trichomes were highly enriched with Ca. Mesophyll cells appeared to have more Zn than the epidermis, probably because the latter were very small in size. Similarities and differences between A. halleri and the other well‐known Zn hyperaccumulator, Thlaspi caerulescens, are discussed.     

Cd speciation and localization in the hyperaccumulator Arabidopsis halleri

Arabidopsis halleri is a model plant investigated for Zn and Cd hyperaccumulation. In this work, A. halleri plants from metallicolous origin exposed to Cd and Zn were studied using an original combination of chemical and physical techniques to obtain insights on Cd hyperaccumulation mechanisms. In most cases, Zn and Cd accumulation in leaves increased with time and with their respective concentrations in hydroponics. At the rosette scale, ¹⁰⁹Cd autoradiography showed that the amount of Cd increased with time but there was no clear trend in the localization of Cd in young or mature leaves. At the leaf scale, an enrichment of the petiole, central vein and trichomes was observed after 3 weeks. After 9 weeks, leaf edges were the most Cd-enriched tissues, and regions along leaf vascular bundles appeared less concentrated. Bulk Cd K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy showed that Cd was predominantly bound to COOH/OH groups belonging to organic acids and/or cell wall components. Cd bound to thiol groups was found as a secondary species (less than 25%). Thiols ligands might correspond, at least partly, to glutathione found in significant amount in aerial parts, but phytochelatins were not detected. These results show that the mechanisms of Cd storage and detoxification in A. halleri differ from what was previously found for Zn.     

Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens

The aim of this study was to show the potential of Thlaspi caerulescens in the cleaning‐up of a moderately Zn ‐contaminated soil and to elucidate tolerance mechanisms at the cellular and subcellular level for the detoxification of the accumulated metal within the leaf. Measured Zn concentrations in shoots were high and reached a maximum value of 83 mmol kg ^{? 1} dry mass, whereas total concentrations of Zn in the roots were lower (up to 13 mmol kg ^{? 1}). In order to visualize and quantify Zn at the subcellular level in roots and leaves, ultrathin cryosections were analysed using energy‐dispersive X‐ray micro‐analysis. Elemental maps of ultrathin cryosections showed that T. caerulescens mainly accumulated Zn in the vacuoles of epidermal leaf cells and Zn was almost absent from the vacuoles of the cells from the stomatal complex, thereby protecting the guard and subsidiary cells from high Zn concentrations. Observed patterns of Zn distribution between the functionally different epidermal cells were the same in both the upper and lower epidermis, and were independent of the total Zn content of the plant. Zinc stored in vacuoles was evenly distributed and no Zn‐containing crystals or deposits were observed. From the elemental maps there was no indication that P, S or Cl was associated with the high Zn concentrations in the vacuoles. In addition, Zn also accumulated in high concentrations in both the cell walls of epidermal cells and in the mesophyll cells, indicating that apoplastic compartmentation is another important mechanism involved in zinc tolerance in the leaves of T. caerulescens.     

Characterization of Cd translocation and identification of the Cd form in xylem sap of the Cd-hyperaccumulator Arabidopsis halleri

Arabidopsis halleri is a Cd hyperaccumulator; however, the mechanismsinvolved in the root to shoot translocation of Cd are not wellunderstood. In this study, we characterized Cd transfer fromthe root medium to xylem in this species. Arabidopsis halleriaccumulated 1,500 mg kg^–1 Cd in the shoot without growthinhibition. A time-course experiment showed that the releaseof Cd into the xylem was very rapid; by 2 h exposure to Cd,Cd concentration in the xylem sap was 5-fold higher than thatin the external solution. The concentration of Cd in the xylemsap increased linearly with increasing Cd concentration in theexternal solution. Cd transfer to the xylem was completely inhibitedby the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone(CCCP). Cd concentration in the xylem sap was decreased by increasingthe concentration of external Zn, but enhanced by Fe deficiencytreatment. Analysis with ¹¹³Cd-nuclear magnetic resonance (NMR)showed that the chemical shift of ¹¹³Cd in the xylem sap wasthe same as that of Cd(NO₃)₂. Metal speciation with Geochem-PCalso showed that Cd occurred mainly in the free ionic form inthe xylem sap. These results suggest that Cd transfer from theroot medium to the xylem in A. halleri is an energy-dependentprocess that is partly shared with Zn and/or Fe transport. Furthermore,Cd is translocated from roots to shoots in inorganic forms.     

Compartmentation of Zinc in Roots and Leaves of the Zinc Hyperaccumulator Thlaspi caerulescens J & C Presl

Thlaspi caerulescens is a metallophyte that is able to hyperaccumulate Zn. In the present study the subcellular compartmentation of Zn was investigated in roots and leaves of this species by means of X-ray microanalysis. Leaves accumulated higher average Zn concentrations than roots. In roots of plants exposed to 10 μM Zn, Zn concentrations in the apoplast were similar to those in vacuoles, while in plants treated with 100 μM Zn considerably higher Zn concentrations were detected in vacuoles than in the apoplast. In epidermal and sub-epidermal cells of leaves of plants from both treatments, Zn mainly accumulated in vacuoles and, to a lesser extent, in the apoplast. In vacuoles from plants exposed to 100 μM Zn, high Zn concentrations were associated with variable amounts of P, Ca and K. In leaves, the highest Zn concentrations (13,600 μg g^?1 d.m.) were found in globular crystals present in many vacuoles of epidermal and subepidermal cells. Smaller deposits with a variable Zn concentration between 1,000 and 18,300 μg g^?1 d.m. were observed in the epidermal and subepidermal cells of roots. Both the high Zn/P element ratios found in the crystals and the absence of Mg indicate that, in contrast to other plant species, myo-inositol hexaphosphate (phytate) is not the main storage form for Zn in Thlaspi caerulescens.     

Morphophysiological responses and programmed cell death induced by cadmium in <Emphasis Type="Italic">Genipa americana</Emphasis> L. (Rubiaceae)

Cadmium (Cd) originating from atmospheric deposits, from industrial residues and from the application of phosphate fertilizers may accumulate in high concentrations in soil, water and food, thus becoming highly toxic to plants, animals and human beings. Once accumulated in an organism, Cd discharges and sets off a sequence of biochemical reactions and morphophysiological changes which may cause cell death in several tissues and organs. In order to test the hypothesis that Cd interferes in the metabolism of G. americana, a greenhouse experiment was conducted to measure eventual morphophysiological responses and cell death induced by Cd in this species. The plants were exposed to Cd concentrations ranging from 0 to 16 mg l⁻¹, in a nutritive solution. In TUNEL reaction, it was shown that Cd caused morphological changes in the cell nucleus of root tip and leaf tissues, which are typical for apoptosis. Cadmium induced anatomical changes in roots and leaves, such as the lignification of cell walls in root tissues and leaf main vein. In addition, the leaf mesophyll showed increase of the intercellular spaces. On the other hand, Cd caused reductions in the net photosynthetic rate, stomatal conductance and leaf transpiration, while the maximum potential quantum efficiency of PS2 (Fv/Fm) was unchanged. Cadmium accumulated in the root system in high concentrations, with low translocation for the shoot, and promoted an increase of Ca and Zn levels in the roots and a decrease of K level in the leaves. High concentrations of Cd promoted morphophysiological changes and caused cell death in roots and leaves tissues of G. americana.     

Cd AND Zn TOLERANCE AND ACCUMULATION BY SEDUM JINIANUM IN EAST CHINA

Field survey, hydroponic culture, and pot experiments were carried out to examine and characterize cadmium (Cd) and zinc (Zn) uptake and accumulation by Sedum jinianum, a plant species native to China. Shoot Cd and Zn concentrations in S. jinianum growing on a lead/Zn mine area reached 103–478 and 4165–8349 mg kg^?1 (DM), respectively. The shoot Cd concentration increased with the increasing Cd supply, peaking at 5083 mg kg^?1 (DM) when grown in nutrient at a concentration of 100 μmol L^?1 for 32 d, and decreased as the solution concentration increased from 200 to 400 μmol L^?1. The shoot-to-root ratio of plant Cd concentrations was > 1 when grown in solution Cd concentrations ≤ 200 μmol L^?1. Foliar, stem, and root Zn concentrations increased linearly with the increasing Zn level from 1 to 9600 μmol L^?1. The Zn concentrations in various plant parts decreased in the order roots > stem > leaves, with maximum concentrations of 19.3, 33.8, and 46.1 g kg^?1 (DM), respectively, when plants were grown at 9600 μmol Zn L^?1 for 32 d. Shoot Cd concentrations reached 16.4 and 79.8 mg kg^?1 (DM) when plants were grown in the pots of soil with Cd levels of 2.4 mg kg^?1 and 9.2 mg kg^?1, respectively. At soil Zn levels of 619 and 4082 mg kg^?1, shoot Zn concentrations reached 1560 and 15,558 mg kg^?1 (DM), respectively. The results indicate that S. jinianum is a Cd hyperaccumulator with a high capacity to accumulate Zn in the shoots.     

Cd、Zn及其交互作用对互花米草中重金属的积累、亚细胞分布及化学形态的影响

通过盆栽试验,研究了Cd、Zn及其交互作用下互花米草中Cd、Zn的含量及积累量,并分析了Cd、Zn在互花米草中的亚细胞分布及化学形态。结果表明：Cd-Zn处理组互花米草地上部及根部Cd含量显著高于Cd处理组;Cd-Zn处理组根部Zn含量显著低于Zn处理组,但地上部差异不显著,说明Zn促进Cd的吸收,Cd抑制Zn的吸收。Cd-Zn处理组互花米草地上部Cd积累量显著高于Cd处理组,但是根部Cd积累量却显著低于Cd处理组;Zn处理组地上部及根部Zn积累量均显著高于对照组及Cd-Zn处理组。Cd单因素胁迫下,Cd主要分布在细胞壁,Cd-Zn交互作用下,Cd在胞液中的分配比例高于其他细胞组分;Zn单因素及Cd-Zn交互作用下,Zn在胞液中的分配比例均较高,总的分配比例呈现以下趋势：胞液>细胞壁>细胞器,说明Zn的添加影响了Cd的亚细胞分布,Cd的出现对Zn在互花米草细胞中的分布影响不明显。Cd和Zn在互花米草叶中主要以氯化钠提取态存在,表明互花米草中Cd和Zn多以果胶酸盐结合态或蛋白质结合态存在。     

Genetic basis of Cd tolerance and hyperaccumulation in Arabidopsis halleri

The genetic basis of Cd tolerance and hyperaccumulation was investigated in Arabidopsis halleri. The study was conducted in hydroponic culture with a backcross progeny, derived from a cross between A. halleri and a non-tolerant and non-accumulating related species Arabidopsis lyrata ssp. petraea, as well as with the parents of the backcross. The backcross progeny segregates for both cadmium (Cd) tolerance and accumulation. The results support that (i) Cd tolerance may be governed by more than one major gene, (ii) Cd tolerance and Cd accumulation are independent characters, (iii) Cd and Zn tolerances co-segregate suggesting that they are under pleiotropic genetic control, at least to a certain degree, (iv) the same result was obtained for Cd and Zn accumulation.     

Different strategies of Cd tolerance and accumulation in Arabidopsis halleri and Arabidopsis arenosa

Pseudometallophytes are commonly used to study the evolution of metal tolerance and accumulation traits in plants. Within the Arabidopsis genus, the adaptation of Arabidopsis halleri to metalliferous soils has been widely studied, which is not the case for the closely related species Arabidopsis arenosa. We performed an in-depth physiological comparison between the A. halleri and A. arenosa populations from the same polluted site, together with the geographically close non-metallicolous (NM) populations of both species. The ionomes, growth, photosynthetic parameters and pigment content were characterized in the plants that were growing on their native site and in a hydroponic culture under Cd treatments. In situ, the metallicolous (M) populations of both species hyperaccumulated Cd and Zn. The NM population of A. halleri hyperaccumulated Cd and Zn while the NM A. arenosa did not. In the hydroponic experiments, the NM populations of both species accumulated more Cd in their shoots than the M populations. Our research suggests that the two Arabidopsis species evolved different strategies of adaptation to extreme metallic environments that involve fine regulation of metal homeostasis, adjustment of the photosynthetic apparatus and accumulation of flavonols and anthocyanins.     

Isolation and characterization of <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> phytochelatin synthases

The synthesis of phytochelatins (PC) represents a major metal and metalloid detoxification mechanism in various species. PC most likely play a role in the distribution and accumulation of Cd and possibly other metals. However, to date, no studies have investigated the phytochelatin synthase (PCS) genes and their expression in the Cd-hyperaccumulating species. We used functional screens in two yeast species to identify genes expressed by two Cd hyperaccumulators (Arabidopsis halleri and Thlaspi caerulescens) and involved in cellular Cd tolerance. As a result of these screens, PCS genes were identified for both species. PCS1 was in each case the dominating cDNA isolated. The deduced sequences of AhPCS1 and TcPCS1 are very similar to AtPCS1 and their identity is particularly high in the proposed catalytic N-terminal domain. We also identified in A. halleri and T. caerulescens orthologues of AtPCS2 that encode functional PCS. As compared to A. halleri and A. thaliana, T. caerulescens showed the lowest PCS expression. Furthermore, concentrations of PC in Cd-treated roots were the highest in A. thaliana, intermediate in A. halleri and the lowest in T. caerulescens. This mirrors the known capacity of these species to translocate Cd to the shoot, with T. caerulescens being the best translocator. Very low or undetectable concentrations of PC were measured in A. halleri and T. caerulescens shoots, contrary to A. thaliana. These results suggest that extremely efficient alternative Cd sequestration pathways in leaves of Cd hyperaccumulators prevent activation of PC synthase by Cd²⁺ ions.     

Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of<Emphasis Type="Italic"> Thlaspi caerulescens</Emphasis>

Thlaspi caerulescens (Ganges ecotype) is able to accumulate large concentrations of cadmium (Cd) and zinc (Zn) in the leaves without showing any toxicity, suggesting a strong internal detoxification. The distribution of Cd and Zn in the leaves was investigated in the present study. Although the Cd and Zn concentrations in the epidermal tissues were 2-fold higher than those of mesophyll tissues, 65–70% of total leaf Cd and Zn were distributed in the mesophyll tissues, suggesting that mesophyll is a major storage site of the two metals in the leaves. To examine the subcellular localisation of Cd and Zn in mesophyll tissues, protoplasts and vacuoles were isolated from plants exposed to 50 M Cd and Zn hydroponically. Pure protoplasts and vacuoles were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. Of the total Cd and Zn in the mesophyll tissues, 91% and 77%, respectively, were present in the protoplast, and all Cd and 91% Zn in the protoplast were localised in the vacuoles. Furthermore, about 70% and 86% of total Cd and Zn, respectively, in the leaves were extracted in the cell sap, suggesting that most Cd and Zn in the leaves is present in soluble form. These results indicate that internal detoxification of Cd and Zn in Thlaspi caerulescens leaves is achieved by vacuolar compartmentalisation.     

利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

The long-term variation of Cd and Zn hyperaccumulation by Noccaea spp and Arabidopsis halleri plants in both pot and field conditions

Three Cd and Zn hyperaccumulating plant species Noccaea caerulescens Noccaea praecox and Arabidopsis halleri (Brassicacceae) were cultivated in seven subsequent vegetation seasons in both pot and field conditions in soil highly contaminated with Cd, Pb, and Zn. The results confirmed the hyperaccumulation ability of both plant species, although A. halleri showed lower Cd uptake compared to N. caerulescens. Conversely, Pb phytoextraction was negligible for both species in this case. Because of the high variability in plant yield and element contents in the aboveground biomass of plants, great variation in Cd and Zn accumulation was observed during the experiment. The extraction ability in field conditions varied in the case of Cd from 0.2 to 2.9 kg ha^?1 (N. caerulescens) and up to 0.15 kg ha^?1 (A. halleri), and in the case of Zn from 0.2 to 6.4 kg ha^?1 (N. caerulescens) and up to 13.8 kg.ha^?1 (A. halleri). Taking into account the 20 cm root zone of the soil, the plants were able to extract up to 4.1% Cd and 0.2% Zn in one season. However, cropping measures should be optimized to improve and stabilize the long-term phytoextraction potential of these plants.     

Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance

The heavy metal zinc was administered to barley seedlings by increasing its concentration in the hydroponic medium. The most dramatic effect was a severe inhibition of root elongation with little effect on root biomass production. The growth of primary leaves was little affected although the zinc content of the primary leaves increased several-fold. A detailed compartment analysis was performed for 10-d-old barley primary leaves. Under low zinc nutrition (2mmol m ⁻³), highest zinc contents were observed in the cytoplasm of mesophyll protoplasts. At inhibitory zinc concentrations in the hydroponic medium (400 μmol m ⁻³), zinc levels dramatically and preferentially increased in the apoplastic space. Elevated zinc levels were also observed in the epidermal cells, and to a lesser extent, in mesophyll vacuoles. The cytoplasmic content of mesophyll protoplasts was unchanged, indicating perfect zinc homeostasis within the leaf. In order to understand the transport mechanisms underlying the steady-state distribution profile, we used ⁶⁵Zn to conduct uptake experiments with leaves whose lower epidermis had been stripped. The leaves were placed on zinc solutions of varying concentrations containing ⁶⁵Zn for 5 min to 6 h. After the incubation, the leaves were fractionated into mesophyll and epidermis protoplasts and residue, the latter mainly representing cell wall. Adsorption of Zn to the extracellular matrix was 100 times faster than Zn uptake into the cells. By far the largest portion taken up into the mesophyll protoplasts rapidly appeared in the vacuolar compartment. These results demonstrate the importance of compartmentation and transport as homeostatic mechanisms within the leaves to handle high, possibly toxic, zinc levels in the shoot.     

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

Grown under saline conditions, Suaeda maritima accumulates Na⁺ and Cl^- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na⁺, K⁺ and Cl^- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.Abbreviation TAEM transmission analytical electron microscopy     

Cadmium tolerance in Thlaspi caerulescens: II. Localization of cadmium in Thlaspi caerulescens

Tissue and cellular compartmentation of Cd in roots and leaves of the hyperaccumulator Thlaspi caerulescens was investigated, using energy-dispersive X-ray microanalysis. In roots, Cd was determined in cortex parenchyma cells, endodermis, parenchyma cells of the central cylinder and xylem vessels. In leaves, it was found in cells lying on the way of water migration from the vascular cylinder to epidermal cells, which is in line with passive Cd transport by the transpiration stream. The mechanisms of Cd-detoxification in roots seem to be localized both in apoplast (e.g. binding to cell wall compounds) and inside cells since Cd was accumulated in these two compartments. On the other hand, in leaves Cd was found only in electron-dense deposits inside vacuoles, which suggests that vacuoles are the main compartment of its storage and detoxification in these organs.     

Phytoextraction of Cd and Zn from agricultural soils by Salix ssp. and intercropping of Salix caprea and Arabidopsis halleri

Contamination of agricultural topsoils with Cd above guideline values is of concern in many countries throughout the world. Extraction of metals from contaminated soils using high-biomass, metal-accumulating Salix sp. has been proposed as a low-cost, gentle remediation strategy, but reasonable phytoextraction rates remain to be demonstrated. In an outdoor pot experiment we assessed the phytoextraction potential for Cd and Zn of four willow species (Salix caprea, S. fragilis, S. × smithiana, S. × dasyclados) and intercropping of S. caprea with the hyperaccumulator Arabidopsis halleri on three moderately contaminated, agricultural soils. Large concentrations of Cd (250 mg kg⁻¹) and Zn (3,300 mg kg⁻¹) were determined in leaves of Salix × smithiana grown on a soil containing 13.4 mg kg⁻¹ Cd and 955 mg kg⁻¹ Zn, resulting in bioaccumulation factors of 27 (Cd) and 3 (Zn). Total removal of up to 20% Cd and 5% Zn after three vegetation periods were shown for Salix × smithiana closely followed by S. caprea, S. fragilis and S. × dasyclados. While total Cd concentrations in soils were reduced by up to 20%, 1 M NH₄NO₃-extractable metal concentrations did not significantly decrease within 3 years. Intercropping of S. caprea and A. halleri partly increased total removal of Zn, but did not enhance total Cd extraction compared to single plantings of S. caprea after two vegetation periods.