Environmental relevance of heavy metal-substituted chlorophylls using the example of water plants

Following experiments which studied the substitution of thecentral ion of isolated chlorophylls by heavy metal ions invitro, in vivo experiments with submersed water plants werecarried out. It was discovered that the substitution of thecentral atom of chlorophyll, magnesium, by heavy metals (mercury,copper, cadmium, nickel, zinc, lead) in vivo is an importantdamage mechanism in stressed plants. This substitution preventsphotosynthetic light-harvesting in the affected chlorophyllmolecules, resulting in a breakdown of photosynthesis. The reactionvaries with light intensity. In low light irradiance all thecentral atoms of the chlorophylls are accessible to heavy metals,with heavy metal chlorophylls being formed, some of which aremuch more stable towards irradiance than Mg-chlorophyll. Consequently,plants remain green even when they are dead. In high light,however, almost all chlorophyll decays, showing that under suchconditions most of the chlorophylls are inaccessible to heavymetal ions. Key words: Heavy metal chlorophylls, submersed water plants, antenna pigments, copper, zinc     

Histochemical methods for detection of heavy metals and strontium in the tissues of higher plants

Elaboration and application of histochemical methods for detection of heavy metals (Cd, Pb, Ni, Zn) and strontium for the purpose of investigating their distribution, accumulation, and translocation within the tissues of higher plants are discussed. Detailed protocols of metal detection with metallochrome indicators dithizone (Cd, Pb), dimethylglyoxime (Ni), sodium rhodizonate (Sr), zincon (Zn), and fluorescent indicator Zinpyr-1 (Zn) by light and fluorescence microscopy are described. Special attention is given to interpretation of the obtained results, advantages and drawbacks of these methods, as well as potential problems associated with histochemical analysis of distribution of heavy metals and strontium.     

Assessing the poplar photochemical response to high zinc concentrations by image processing and statistical approach

Exposure of plants to high-heavy metals concentration inhibits multiple metabolic processes in plants and leads to an oxidative stress commonly referred as heavy metal ion toxicity. Chlorophyll a fluorescence has enhanced understanding of heavy metal ion action on the photosynthetic system. A rapid and non-invasive technique involving imaging of chlorophyll fluorescence is a useful tool for early detection of plant responses to heavy metal ion toxicity. In this work chlorophyll fluorescence emission and photochemical parameters in plants of Populus x euramericana clone I-214 were investigated by the portable Imaging PAM fluorometer at different days after soil treatment with zinc. Custom software for analysis of the photochemical parameters images has been developed in order to gain a better assessing of the plant performance in response of metal stress. The imaging analysis allowed visualizing heterogeneity in plant response to high zinc concentrations. The heterogeneity of images suggests spatial differences in photochemical activity and changes in the antenna down-regulation.     

Development of surface‐engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene‐excimer fluorescence

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface‐engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level‐dependent synthesis of PC₂ from glutathiones by AtPCS1‐displaying yeast cells, followed by simple discriminative detection of PC₂ via sensing of excimer fluorescence of thiol‐labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose‐dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross‐reaction, the proposed simple biosensor holds promise as a method useful for cost‐effective screening of Cd contamination in environmental and food samples. The AtPCS1‐displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1197–1202, 2013     

Cd2+胁迫对银芽柳PSⅡ叶绿素荧光光响应曲线的影响

以盆栽银芽柳为材料,利用MINI-IMAGING-PAM荧光成像测定系统,研究了Cd²⁺胁迫下叶片叶绿素荧光参数的变化及其光响应曲线。结果表明,初始荧光Fo与最大荧光Fm随着Cd²⁺浓度的增大而呈现先升后降的趋势,Fo与Fm在200 mg/LCd²⁺处理4周时达到最高值,400 mg/LCd²⁺处理则显著下降;PSⅡ最大光化学效率(Fv/Fm)与PSⅡ潜在光化学效率(Fv/Fo)显著受 Cd²⁺胁迫抑制,但随Cd²⁺浓度的增加呈先降后升的变化趋势。Cd²⁺胁迫下各叶绿素荧光参数的光响应结果表明,PSⅡ实际光量子效率Y(Ⅱ)、荧光淬灭系数(qP)随光化光强度的增加呈下降趋势,而同光强下高浓度Cd²⁺ 使Y(Ⅱ)与(qP) 显著降低;PSⅡ调节性能量耗散的量子产额Y(NPQ)、非光化学淬灭系数(qN)与表观电子传递速率(ETR)则随着光强增加呈上升趋势,同光强下高浓度Cd²⁺处理显著提高Y(NPQ)、qN 与ETR。Cd²⁺胁迫下,PSⅡ非调节性能量耗散的量子产额Y(NO)稳定在较低水平,同光强下Y(NO)随Cd²⁺浓度增加略有提高。说明,银芽柳通过调节PSⅡ反应中心开放程度与活性,对Cd²⁺胁迫表现出较强的耐性,高浓度Cd²⁺胁迫导致PSⅡ反应中心关闭或不可逆失活,表现出光抑制。     

Inhibition of photosynthesis by heavy metals

Inhibition of photosynthesis by heavy metals is well documented. In this review the results are compared between in vitro experiments on isolated systems (chloroplasts, enzymes ­.), experiments on excised leaves and intact plants and algae in vivo. In vitro experiments suggest potential sites of heavy metal interaction with photosynthesis at several levels of organisation, which are not necessarily confirmed in vivo. Analytical data on subcellular heavy metal level are generally missing to discuss their mechanism of action in the intact organism. In the field factors such as soil characteristics and air pollution have to be taken into account for assessing the mechanism of action of heavy metals on photosynthesis in plants, growing in a polluted erea.paper presented at the FESPP meeting in Strasbourg (1984)     

AM真菌对重金属污染土壤生物修复的应用与机理

土壤重金属污染威胁人类健康和整个生态系统,而高效、低耗、安全的生物修复技术显示出了极大的应用潜力,特别是利用植物-微生物共生体增强生物修复效应的应用.丛枝菌根(Arbuscular Mycorrhizae,AM)真菌是一类广泛分布于土壤生态系统中的有益微生物,能与90％以上的陆生高等植物形成共生体.研究发现,AM真菌能够增强宿主植物对土壤中重金属胁迫的耐受性.当前,利用AM真菌开展重金属污染土壤的生物修复已经引起环境学家和生态学家的广泛关注.基于此,围绕AM真菌在重金属污染土壤生物修复作用中的最新研究进展,从物理性防御体系的形成、对植物生理代谢的调控、生化拮抗物质的产生、基因表达的调控等角度探究AM真菌在重金属污染土壤生物修复中的作用机理,以期为利用AM真菌开展重金属污染的生物修复提供理论依据,并对本领域未来的发展和应用前景进行了展望.     

Heavy metals toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants

Plants experience oxidative stress upon exposure to heavy metals that leads to cellular damage. In addition, plants accumulate metal ions that disturb cellular ionic homeostasis. To minimize the detrimental effects of heavy metal exposure and their accumulation, plants have evolved detoxification mechanisms. Such mechanisms are mainly based on chelation and subcellular compartmentalization. Chelation of heavy metals is a ubiquitous detoxification strategy described in wide variety of plants. A principal class of heavy metal chelator known in plants is phytochelatins (PCs), a family of Cys-rich peptides. PCs are synthesized non-translationally from reduced glutathione (GSH) in a transpeptidation reaction catalyzed by the enzyme phytochelatin synthase (PCS). Therefore, availability of glutathione is very essential for PCs synthesis in plants at least during their exposure to heavy metals. Here, I reviewed on effect of heavy metals exposure to plants and role of GSH and PCs in heavy metal stress tolerance. Further, genetic manipulations of GSH and PCs levels that help plants to ameliorate toxic effects of heavy metals have been presented.     

磷酸盐修复重金属污染土壤的研究进展

从研究方法、反应机理以及风险评价等方面综述了磷酸盐修复重金属污染土壤的研究进展,分析和讨论了其中存在的问题和不足,提出了今后加强研究的重点。目前磷酸盐修复重金属污染土壤时,使用的主要研究方法有化学形态提取法、化学平衡形态模型法和光谱及显微镜技术,各个方法都有其优缺点,应该结合使用并探索新方法。磷酸盐稳定重金属的作用机理主要有3个:磷酸盐诱导重金属吸附、磷酸盐和重金属生成沉淀或矿物和磷酸盐表面吸附重金属,但磷酸盐与重金属反应的机理十分复杂,人们尚不完全清楚,因此难以有效区分和评价诱导吸附机理和沉淀机理或其它固定机理,相应地对磷酸盐修复重金属的长期稳定性难以预测。磷酸盐修复重金属污染土壤时由于其较高的施用量可能会造成磷的积聚从而引发一些环境风险,如磷淋失造成水体富营养化,营养失衡造成作物必需的中量和微量元素缺乏以及土壤酸化等。所以应该谨慎选择磷肥种类和用量,最好是水溶性磷肥和难溶性磷肥配合、磷肥与石灰物质等配合施用。今后应着重研究磷酸盐与重金属相互作用的机理区分和评价;关注磷酸盐修复重金属污染土壤时存在的潜在风险,特别是加强植物长期不断吸收磷或其它环境条件变化致使土壤磷素持续减少过程中稳定的重金属溶解性和移动性的研究,磷酸盐修复重金属污染土壤的长期田间实践等。     

Managing heavy metal toxicity stress in plants: Biological and biotechnological tools

The maintenance of ion homeostasis in plant cells is a fundamental physiological requirement for sustainable plant growth, development and production. Plants exposed to high concentrations of heavy metals must respond in order to avoid the deleterious effects of heavy metal toxicity at the structural, physiological and molecular levels. Plant strategies for coping with heavy metal toxicity are genotype-specific and, at least to some extent, modulated by environmental conditions. There is considerable interest in the mechanisms underpinning plant metal tolerance, a complex process that enables plants to survive metal ion stress and adapt to maintain growth and development without exhibiting symptoms of toxicity. This review briefly summarizes some recent cell biological, molecular and proteomic findings concerning the responses of plant roots to heavy metal ions in the rhizosphere, metal ion-induced reactions at the cell wall-plasma membrane interface, and various aspects of heavy metal ion uptake and transport in plants via membrane transporters. The molecular and genetic approaches that are discussed are analyzed in the context of their potential practical applications in biotechnological approaches for engineering increased heavy metal tolerance in crops and other useful plants.     

Effect of the Heavy Metals on Developmental Stages of Ovule,Pollen, and Root Proteins in <Emphasis Type="Italic">Reseda lutea</Emphasis> L. (Resedaceae)

Heavy metals are some of the most important environmental pollutants. Excessive amounts of heavy metals adversely affect plant growth and development. Also, the presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. The overall objective of this research was to elucidate some microscopic effects of heavy metals on the formation, development, and structure of pollen, ovule, and embryo and also root proteins in Reseda lutea L. For this purpose, the vicinity of Ahangaran lead–zinc mine (Hamedan, Iran) was chosen as a polluted area where amount of some heavy metals was several times higher than the natural soils. Flowers and young buds were collected from non-polluted and polluted plants, fixed in FAA₇₀, and studied during developmental stages by light microscopy. The results showed that heavy metals can cause some abnormalities during the pollen and ovule developmental process. The number of pollen grains was decreased, and their shape was changed. Increasing in thickness of the callosic wall and stabilizing of tapetum layer were observed in polluted plants. Asymmetrical formation of ovular integuments, degradation of egg apparatus, irregular formation of embryo sac, considerable vacuolation of embryonic cells, and degeneration of embryo in the late stage of heart-shaped embryo are the results of heavy metal pollution. For protein studies, young roots were harvested from plants exposed to pollution and non-exposed to pollution at the same time. Root proteins were extracted and studied by electrophoresis. The results revealed that some new proteins were synthesized in polluted samples that probably elevate plant tolerance to heavy metals.     

The potential of phytoremediation using hyperaccumulator plants: a case study at a lead-zinc mine site

Contamination with heavy metals is one of the most pressing threats to water and soil resources, as well as human health. Phytoremediation might potentially be used to remediate metal-contaminated sites. A major advance in the development of phytoremediation for heavy metal affected soils was the discovery of heavy metal hyperaccumulation in plants. This study applied several established criteria to identify hyperaccumulator plants. A case study was conducted at a mining area in the Hamedan province in the west central region of Iran. The results indicated that plant metal accumulation differed among species and plant parts. Plant species grown in substrata with elevated metal levels contained significantly higher metal levels. Using the most common criteria, Euphorbia macroclada and Centaurea virgata can be classified as hyperaccumulators of specific heavy metals measured in this study and they might potentially be used for the phytoremediation of contaminated soils.     

植物螯合肽及其在重金属耐性中的作用

综述植物螯合肽的生物合成及其在重金属耐性中的作用．有毒重金属在土壤中的积累不仅影响作物的生长和产量形成,而且严重威胁农产品的安全性．植物对重金属的耐性和积累在种间和基因型之间存在着很大的差异,在重金属胁迫条件下植物螯合肽(PC)的合成是植物对胁迫的一种适应性反应,耐性基因型合成较多的PC谷胱苷肽是合成PC的前体,PC可与重金属螯合,并进一步转运至液泡贮存,使细胞质的重金属浓度降低,从而达到解毒效果．重金属诱导植物合成PC的遗传机理和生化途径有赖于分子生物学的深入研究,cD-敏感型拟南芥突变体Cad1-1(缺失GSH)和Cad2-1(缺失PC合成诱导酶)的分离及相关研究,佐证了PC在Cd-解毒中起关键作用．对PC在重金属污染土壤或水体的植物修复和农作物安全生产中的意义进行了讨论．     

大蒜植物络合素合酶基因转化对酵母重金属抗性的提高

重金属污染是全球面临的亟待解决的生态问题。利用植物对重金属的富集作用来清除环境重金属污染即植物修复已成为重要的环境生物技术之一。这一技术的长远发展有赖于在重金属富集或耐受中起关键作用的基因的克隆和应用。植物络合素是植物体内一类重要的对重金属起螯合作用的多肽, 其合成受植物络合素合酶的催化。该文取得了如下研究结果:1)通过原子吸收测定表明,在大蒜(Allium sativum)的根部可以积累3 000 mg·kg^-1的重金属镉;2)将克隆的大蒜植物络合素合酶基因(AsPCS)置于酵母表达启动子之下,构建酵母表达载体,并将其分别转入了因CUP1和acr3基因缺失而对重金属镉和砷敏感的酵母突变体菌株后,发现来自大蒜的AsPCS基因的表达使酵母CUP1缺失菌株对镉的耐受性提高了4倍, acr3缺失菌株对砷的耐受性提高了两倍;3)表达AsPCS基因酵母的生长模式证实了AsPCS基因的表达是酵母对重金属耐受性提高的原因。这些结果暗示, 大蒜植物络合素合酶基因在大蒜对重金属的抗性及大蒜根部对镉的积累中起关键作用,可作为重要的基因元件应用到修复污染的植物基因工程中。     

Optimizing phytoremediation of heavy metal-contaminated soil by exploiting plants' stress adaptation

Soil phytoextraction is based on the ability of plants to extract contaminants from the soil. For less bioavailable metals, such as Pb, a chelator is added to the soil to mobilize the metal. The effect can be significant and in certain species, heavy metal accumulation can rapidly increase 10-fold. Accumulation of high levels of toxic metals may result in irreversible damage to the plant. Monitoring and controlling the phytotoxicity caused by EDTA-induced metal accumulation is crucial to optimize the remedial process, i.e. to achieve maximum uptake. We describe an EDTA-application procedure that minimizes phytotoxicity by increasing plant tolerance and allows phytoextraction of elevated levels of Pb and Cd. Brassica juncea is tested in soil with typical Pb and Cd concentrations of 500 mg kg-1 and 15 mg kg-1, respectively. Instead of a single dose treatment, the chelator is applied in multiple doses, that is, in several small increments, thus providing time for plants to initiate their adaptation mechanisms and raise their damage threshold. In situ monitoring of plant stress conditions by chlorophyll fluorescence recording allows for the identification of the saturating heavy metal accumulation process and of simultaneous plant deterioration.     

Barley HvHMA1 Is a Heavy Metal Pump Involved in Mobilizing Organellar Zn and Cu and Plays a Role in Metal Loading into Grains

Heavy metal transporters belonging to the P_1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P_1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

植物对重金属的吸收和分布

植物修复是利用植物来清除污染土壤中重金属的一项技术。该技术成功与否取决于植物从土壤中吸取金属以及向地上部运输金属的能力。植物对金属的吸收主要取决于自由态离子活度。许多螯合剂能诱导植物对重金属的吸收。金属离子在液泡中的区域化分布是植物耐重金属的主要原因。同时,细胞内的金属硫蛋白、植物螯合脓等蛋白质以及有机酸、氨基酸等在金属贮存和解毒方面也起重要作用。本文还论述了重金属在植物体内运输的生理及分子方面的研究进展。     

植物对重金属的吸收和分布

植物修复是利用植物来清除污染土壤中重金属的一项技术。该技术成功与否取决于植 物从土壤中吸取金属以及向地上部运输金属的能力。植物对金属的吸收主要取决于自由态离子活度。许多螯合剂能诱导植物对重金属的吸收。金属离子在液泡中的区域化分布是植物耐 重金属的主要原因。同时,细胞内的金属硫蛋白、植物螯合肽等蛋白质以及有机酸、氨基酸等在金属贮存和解毒方面也起重要作用。本文还论述了重金属在植物体内运输的生理及分子 方面的研究进展。     

Histochemical assay to detect class III ADH activity in situ in Arabidopsis seedlings.

Whole-mount detection methods are quick, inexpensive and offer the possibility of studying the temporal and spatial patterns of gene expression in a morphological context. These methods have been used widely to detect messenger RNAs and to measure enzymatic activity of reporter genes, such as beta-galactosidase or beta-glucuronidase. Taking advantage of the fact that NADH generated during the oxidation of formaldehyde by class III alcohol dehydrogenase can reduce the compound nitroblue tetrazolium to form a blue precipitate, we have developed a new method to detect class III alcohol dehydrogenase activity in situ in whole Arabidopsis plants. This reaction has been used earlier for in situ electrophoresis detection and for histochemical analysis in animal tissue sections. With a few modifications, it can be used in whole Arabidopsis plants or excised plant tissues to allow a rapid analysis of class III ADH activity during development or in response to elicitors. The method might be extended to other dehydrogenases by using specific substrates.