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.     

Tolerance,accumulation and distribution of zinc and cadmium in hyperaccumulator Potentilla griffithii

In this study, zinc (Zn) and cadmium (Cd) tolerance, accumulation and distribution was conducted in Potentilla griffithii H., which has been identified as a new Zn hyperaccumulator found in China. Plants were grown hydroponically with different levels of Zn²⁺ (20, 40, 80 and 160 mg L^?1) and Cd²⁺ (5, 10, 20 and 40 mg L^?1) for 60 days. All plants grew healthy and attained more biomass than the control, except 40 mg L^?1 Cd treatment. Zn or Cd concentration in plants increased steadily with the increasing addition of Zn or Cd in solution. The maximum metal concentrations in roots, petioles and leaves were 14,060, 19,600 and 11,400 mg kg^?1 Zn dry weight (DW) at 160 mg L^?1 Zn treatment, and 9098, 3077 and 852 mg kg^?1 Cd DW at 40 mg L^?1 Cd treatment, respectively. These results suggest that P. griffithii has a high ability to tolerate and accumulate Cd and Zn, and it can be considered not only as Zn but also as a potential cadmium hyperaccumulator. Light microscope (LM) with histochemical method, scanning electron microscope combined with energy dispersive spectrometry (SEM-EDS) and transmission electron microscope (TEM) were used to determine the distribution of Zn and Cd in P. griffithii at tissue and cellular levels. In roots, SEM-EDS confirmed that the highest Zn concentration was found in xylem parenchyma cells and epidermal cells, while for Cd, a gradient was observed with the highest Cd concentration in rhizodermal and cortex cells, followed by central cylinder. LM results showed that Zn and Cd distributed mainly along the walls of epidermis, cortex, endodermis and some xylem parenchyma. In leaves, Zn and Cd shared the similar distribution pattern, and both were mostly accumulated in epidermis and bundle sheath. However, in leaves of 40 mg L^?1 Cd treatment, which caused the phytotoxicity, Cd was also found in the mesophyll cells. The major storage site for Zn and Cd in leaves of P. griffithii was vacuoles, to a lesser extent cell wall or cytosol. The present study demonstrates that the predominant sequestration of Zn and Cd in cell walls of roots and in vacuoles of epidermis and bundle sheath of leaves may play a major role in strong tolerance and hyperaccumulation of Zn and Cd in P. griffithii.     

Compartmentalization and ultrastructural alterations induced by chromium in aquatic macrophytes

The aim of the present study was to identify the sites of accumulation of Cr in the species of macrophytes that are abundant in the Cachoeira river, namely, Alternanthera philoxeroides, Borreria scabiosoides, Polygonum ferrugineum and Eichhornia crassipes. Plants were grown in nutritive solution supplemented with 0.25 and 50 mg l^?1 of CrCl₃·6H₂O. Samples of plant tissues were digested with HNO₃/HCl in a closed-vessel microwave system and the concentrations of Cr determined using inductively-coupled plasma mass spectrometry (ICP-MS). The ultrastructure of root, stem and leaf tissue was examined using transmission electron microscopy (TEM) and secondary ion mass spectrometry (SIMS) in order to determine the sites of accumulation of Cr and to detect possible alterations in cell organelles induced by the presence of the metal. Chromium accumulated principally in the roots of the four macrophytes (8.6?C30 mg kg^?1 dw), with much lower concentrations present in the stems and leaves (3.8?C8.6 and 0.01?C9.0 mg kg^?1 dw, respectively). Within root tissue, Cr was present mainly in the vacuoles of parenchyma cells and cell walls of xylem and parenchyma. Alterations in the shape of the chloroplasts and nuclei were detected in A. philoxeroides and B. scabiosoides, suggesting a possible application of these aquatic plants as biomarkers from Cr contamination.     

Subcellular localization of chromium and nickel in root cells of Allium cepa by EELS and ESI

The ultrastructural investigation of the root cells of Allium cepa L. exposed to two different concentrations of chromium + nickel (Cr+Ni) (10 micromol/L and 100 micromol/L) revealed that toxic symptoms were induced by increasing heavy metal concentration and treatment time. Several significant ultrastructural changes were caused by 100 micromol/L Cr+Ni - deposition of electron dense material in cell walls; larger vacuolar precipitates surrounded by membranes inside vacuoles; increment of disintegrated organelles and high vacuolization in cytoplasm. The localization of the precipitates in which the metal ions were detected by electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) was investigated. Chromium and nickel were localized in the electron dense precipitates of the root cells exposed to only 100 micromol/L Cr+Ni. None were found in the root cells exposed to 10 micromol/L Cr+Ni. Higher amounts of Cr+Ni were mainly accumulated in the cell walls and vacuoles of the fourth or fifth cortical layer.     

Chromium VI Induced Structural and Ultrastructural Changes in Bush Bean Plants (Phaseolus vulgaris L.)

The effect of a growth reducing chromium VI concentration (9.6x 10^–5M Cr as Na₂CrO₄) on the structure and ultrastructureof different organs of bush bean plants (Phaseolus vulgarisL.) grown on perlite was studied using light, transmission electronand scanning electron microscopy. The structural and ultrastructuralalterations observed were quite different in the various organsanalysed and did not always stay in direct relation to the averageCr content of the whole organ. The primary toxicity effect seemedto be membrane damage, due to the high oxidation power of CrVI. It is suggested that chromium is retained in vacuoles andcell walls of roots, and that the chromium reaching the leavesmay be principally Cr III and present in cell walls. The alterationsobserved in the upper plant parts seemed principally due toindirect Cr effects on the content of essential mineral nutrients. Phaseolus vulgaris L., bush bean, chromium VI toxicity, structure, ultranstructure, light microscopy, transmission electron microscopy, scanning electron microscopy     

SPECTROSCOPIC DETERMINATION OF THE TOXICITY,ABSORPTION, REDUCTION,AND TRANSLOCATION OF Cr(VI) IN TWO MAGNOLIOPSIDA SPECIES

Hexavalent chromium is a contaminant highly mobile in the environment that is toxic for plants at low concentrations. In this work, the physiological response of Convolvulus arvensis and Medicago truncatula plants to Cr(VI) treatments was compared. C. arvensis is a potential Cr hyperaccumulator well adapted to semiarid conditions that biotransform Cr(VI) to the less toxic Cr(III). M. truncatula is a model plant well adapted to semiarid conditions with a well studied genetic response to heavy metal stress. The results demonstrated that C. arvensis is more tolerant to Cr toxicity and has a higher Cr translocation to the leaves. The inductively coupled plasma optical emission spectroscopy results showed that C. arvensis plants treated with 10 mg Cr(VI) L^–1 accumulated 1512, 210, and 131 mg Cr kg^–1 in roots, stems, and leaves, respectively. While M. truncatula plants treated with the same Cr(VI) concentration accumulated 1081, 331, and 44 (mg Cr kg^–1) in roots, stems, and leaves, respectively. Enzymatic assays demonstrated that Cr(VI) decreased ascorbate peroxidase activity and increased catalase activity in M. truncatula, while an opposite response was found in C. arvensis. The x-ray absorption spectroscopy studies showed that both plant species reduced Cr(VI) to the less toxic Cr(III).     

Sub-cellular localization of Ni in the hyperaccumulator, Hybanthus floribundus (Lindley) F. Muell

The cellular and subcellular distribution of Ni within leaves of Hybanthus floribundus (Lindley) F. Muell, a hyperaccumulator of Ni, was investigated at relatively high spatial resolution using energy‐dispersive X‐ray microanalysis (EDAX). Elemental distribution maps showed that Ni was predominantly localized in the vacuoles of epidermal cells in the leaves. Quantification of Ni revealed concentrations up to 275 mmol kg^?1 (embedded tissue) in some epidermal vacuoles. The accumulation of Ni in these cells was associated with a decrease in the concentration of Na and K. There was no indication that Ni was associated with P, S or Cl in the vacuoles. Ni was also concentrated on the outside of cell walls throughout the leaves, indicating that apoplastic compartmentation is also involved in Ni tolerance and accumulation in this plant.     

Substitution of Na+ by K+ in Tissues and Root Vacuoles of Barley (Hordeum vulgare L. cv. Aramir)

The aim of this paper was to investigate whether Na⁺ accumulated in the vacuoles of barley roots can be replaced by K⁺, or else remain irreversibly trapped.Barley seedlings (Hordeum vulgare L. cv. Aramir) were grown for 2 days on nutrient solutions and then salinized over 24 h by a linear increase in NaCl concentration (50 mol · m⁻³). NaCl was replaced by an equimolar concentration of KCl and 1 day later the KCl concentration was gradually decreased over 23 hours. After this the plants were grown for 2 further days on basic nutrient solution. Plant material was harvested at each experimental step in order to measure plant growth and K⁺ and Na⁺ concentrations in vacuoles and tissues.The results from quantitated, energy dispersive X-ray analyses on frozen, hydrated vacuoles of the rhizodermls and 3 subsequent cortical cell rows, point to a proportional replacement of K⁺ by Na⁺ after NaCl stress. A replacement of Na⁺ by K⁺ was clearly found in the rhizodermal vacuoles after KCl treatment. Flame emission spectrometric data from corresponding root segments support these results.     

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.     

Cadmium distribution and microlocalization in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) after long-term growth on cadmium-contaminated soil

Brassica napus (L.) was grown from seeds on a reconstituted soil contaminated with 100 mg Cd kg(-1). Compared with roots and stems, leaves accumulated high amounts of Cd. Although the Cd concentration in the leaves remained high throughout plant growth and no appreciable change was noticed in the total, extractable or soluble Cd in the soil adhering to the roots, the symptoms of Cd toxicity (leaf chlorosis, growth retardation) decreased with time. Cd induced a noticeable accumulation of phytochelatins in young plants (aged 22 days), which decreased in parallel to the disappearance of the symptoms of Cd intoxication. The subcellular distribution of Cd in leaves of Cd-acclimated plants was determined using biochemical, microscopic and metal-imaging techniques. Leaf fractionation by differential centrifugations showed that Cd was present predominantly in the 'soluble' fraction corresponding to the vacuoles and the cytoplasm. Transmission electron microscopic analyses revealed that those cell compartments contained electron-dense granules associated with needle-like structures. Cd, and also high amounts of sulfur, was detected in those structures by electron-spectroscopic imaging. This technique also showed Cd binding to cell walls by a mechanism that does not involve sulfur atoms. In contrast, very little Cd was found in chloroplasts, and this is consistent with the preservation of photosynthesis in plants grown on Cd-polluted soil. The microanalytical results presented here confirm that long-term growth of B. napus on Cd-contaminated soil is accompanied by preferential storage of Cd in the vacuoles and the cell walls. This phenomenon diverted Cd ions from metabolically active compartments (cytosol, chloroplasts, mitochondria), resulting in a reduction of Cd toxicity in the leaves.     

The effect of pre-incubation of Allium cepa L. roots in the ATH-rich extract on Pb uptake and localization

The positive influence of anthocyanin (ATH) on toxic metal-treated plant material is well documented; however, it is still not explained if it is caused by changes in element absorption and distribution. Therefore, detailed analysis of the effect of the ATH-rich extract from red cabbage leaves on Pb uptake and localization at morphological, anatomical and ultrastructural level was the goal of this study. Two-day-old adventitious roots of Allium cepa L. (cv. Polanowska) were treated for 2 h with the aqueous solution of Pb(NO₃)₂ at the concentration of 100 μM with or without preliminary incubation in the anthocyanin-rich extract from Brassica oleracea L. var. capitata rubra leaves (250 μM, 3 h). The red cabbage extract did not change the total Pb uptake but it enhanced the translocation of accumulated metal from roots to shoots. Within the pretreated roots, more Pb was deposited in their basal part and definitely smaller amount of the metal was bound in the apoplast of the outer layers of cortex cells. The ultrastructural analysis (transmission electron microscopy and X-ray microanalysis) revealed that the ATH-rich extract lowered the number of Pb deposits in intracellular spaces, cell wall and cytoplasm of root meristematic cells as well as in such organelles important to cell metabolism as mitochondria, plastids and nucleus. The Pb deposits were preferably localised in those vacuoles where ATH also occurred. This sequestration of Pb in vacuoles is probably responsible for reduction of metal cytotoxicity and consequently could lead to better plant growth.     

IMPROVEMENT OF CR PHYTOREMEDIATION BY PISTIA STRATIOTES IN PRESENCE OF NUTRIENTS

The effects of different concentrations of P and N, added separately or combined, on the Cr(III) accumulation capacity of P. stratiotes were studied. Plants and pond water with the addition of contaminant(s) were placed in plastic aquaria. Cr concentration was 5 mg L^–1, while P and N concentrations were 5 mg L^–1or 10 mg L^–1. Nutrient addition significantly favoured Cr removal and enhanced Cr translocation to leaves. In Cr treatments a high detritus formation from loss of root biomass was observed probably due to its toxicity. Cr was mainly accumulated in the detrital fraction, whereas P and N were retained fundamentally in leaves. A toxic effect was observed in the Cr + P10 and Cr + N10 treatments. These results could be applied to enhance Cr removal efficiency of constructed wetlands using P. stratiotes, where nutrient enrichment could be attained by treating sewage together with the industrial effluents.     

Genotypic variation of the responses to chromium toxicity in four oilseed rape cultivars

Heavy metal toxicity in soils has been considered as major constraints for oilseed rape (Brassica napus L.) production. In the present study, toxic effects of chromium (Cr) were studied in 6-d-old seedlings of four different cultivars of B. napus (ZS 758, Zheda 619, ZY 50, and Zheda 622). The elevated content of Cr inhibited seedling growth, decreased the content of photosynthetic pigments, and activities of antioxidant enzymes, as well as increased the content of malondialdehyde and reactive oxygen species in all the cultivars. The Cr content in different parts of plants was higher in Zheda 622 than in other cultivars. The electron microscopic study showed changes in ultrastructure of leaf mesophyll and root tip cells at 400 μmol Cr, and these changes were more prominent in Zheda 622. An increased size and number of starch grains and number of plastoglobuli, damaged thylakoid membranes, and immature nucleoli and mitochondria were observed in leaves. In roots, enlarged vacuoles, disrupted cell walls and cell membranes, an increased number of mitochondria and a size of nucleolus, as well as plasmolysis (in Zheda 622) were observed. On the basis of these findings, it can be concluded that cv. Zheda 622 was more sensitive to Cr as compared to other three cultivars.     

Boron uptake by excised barley roots: I. Uptake into the free space

At 2 C, all boron accumulated by excised barley roots (Hordeum vulgare L. cv. Herta) remains in the free space; i.e. active uptake is nil at this temperature. Three component fractions of free space B were apparent: (a) a surface contaminant film of B on blotted roots, (b) water free space B, and (c) B reversibly bound in the cell walls. A stoichiometric release of H⁺ from the roots in the presence of B indicated that B was bound by borate complexes with polysaccharides in the cell walls. Polysaccharide-borate complexes are much less stable than those of monosaccharides, and the bound B fraction could be readily removed by rinsing the roots in the presence of a monomeric polyol possessing the necessary cis-diol configuration. Cell wall material separated from excised barley roots had a B binding capacity 66% greater than that of intact roots.     

Effect of fluchloralin on protein synthesis, free amino acids and hydroxyproline content in groundnut (Arachis hypogaea L.)

Cultivar TMV-2 of groundnut plant {Arachis hypogaea L.) was grown in a nutrient solution containing fluchloralin at the rate of either 2 mg litre^-1 or 4 mg litre“¹. Protein synthesis and hydroxyproline content in the cell walls of roots, stem and leaves were determined. Free amino acids content and total ammonia in leaves and roots were also analysed. Presence of fluchloralin did not adversely affect protein synthesis. No significant effect of herbicide was observed on hydroxyproline content of a purified cell wall fraction of groundnut roots, stem and leaves. The total amount of ammonia increased in roots and leaves of plants which received the higher concentration of fluchloralin. With the exception of aspartic acid, asparagine, glutamic acid and glutamine, free amino acids content decreased considerably with herbicide treatment. Alanine and glycine were strongly reduced. It is suggested that transamination reactions could be affected and the process of senescence may be enhanced.     

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     

Interactive effects of chromate and arsenate on their uptake and speciation in Pteris ensiformis

Background and aims

Arsenate (AsV) and chromate (CrVI) inhibit each other’s uptake and translocation in As-hyperaccumulator Pteris vittata. In the present study, we extended the research to As-sensitive plant Pteris ensiformis to better understand the mechanism of their interactions.

Methods

Plants were exposed to 0, 0.75 or 7.5 mg L^?1 AsV and 0, 0.52, or 5.2 mg L^?1 CrVI for 7 d in hydroponics. Arsenic and Cr speciation were determined in nutrient solutions and plant biomass.

Results

P. ensiformis accumulated high levels of As and Cr in the rhizomes and roots with low levels in the fronds. However, P. ensiformis was much more effective in taking up Cr than As, as much more Cr was accumulated in the roots (306–6015 vs. 87–642 mg kg^?1). AsV and CrVI increased each other’s uptake in the rhizomes and roots when co-present. The AsV and CrVI taken up by P. ensiformis were reduced to arsenite (AsIII) and chromite (CrIII), possibly serving as detoxification mechanism.

Conclusions

Uptake of As and Cr induced oxidative stress as indicated by increased lipid peroxidation and electrical conductivity. Arsenic and Cr increased each other’s uptake by P. ensiformis.

     

Effects of salinity and temperature stress on Ecophysiological characteristics of exotic cordgrass, Spartina alterniflora

Salintiy and temperature are two important ecological factors which affect the distribution and abundance of Spartina alerniflora Loisel. To find out how cordgrass adapts to the environmental conditions in the introduced range, we studied the dynamics of a series of important physiological components including superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), malondialdehyde (MDA), soluble sugar and free proline under different salinity and temperature stresses. The results showed that low NaCl concentration (lower than 100 mmol·L⁻¹) enhanced the growth of S. alterniflora. On the contrary, high NaCl concentration (higher than 100 mmol·L⁻¹) inhibited the growth of S. alterniflora. To a certain extent, S. alterniflora was able to be acclimated to the osmotic pressure created by external solution concentration by adjusting the activities of POD, SOD and CAT, and the contents of free proline and soluble sugar. S. alterniflora varied in its responses to environment in different parts of the plant under 5°C and 38°C temperature stress. Compared with roots, leaves accumulated more soluble sugar, and CAT activities in leaves were higher, whereas SOD and POD activities in leaves were much lower than those in roots.     

Simulated hypogravity and proline incorporation into salt-extractable macromolecules from cell walls

Proline [U-¹⁴C] was fed to shoots of intact Tagetes patula grown normally, on horizontal clinostats, or on vertical clinostats rotating at 15 rev/hr. After various periods the incorporation of ¹⁴C into salt-extractable material from the cell walls of stems, petioles, leaves and flowers was determined. The cell walls of the gravity-compensated plants (grown on horizontal clinostats) has the highest amount of salt-extractable radioactivity. A 2- to 9-fold increase was observed in comparison to either the normal or vertical clinostat plant controls. Some physico-chemical properties of the salt-extractable fraction suggest that it consists of highly charged, low MW entities, possibly short chain peptides. On acid hydrolysis this material yields radioactive aspartic acid, glutamic acid and proline. The presence of labelled hydroxyproline is suggested. After acid hydrolysis of the cell walls of leaves, it was found that ca 4 times the amount of ¹⁴C was incorporated in the hypogravity-grown plant compared to the controls. It appears likely that extensibility changes in tissues under simulated hypogravity required additional cell wall protein.     

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.