Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metallothionein gene (CUP1)

Cauliflower (Brassica oleracea var. botrytis) tolerates treatment with 25 µM CdCl2 for eight days, but is killed by that with a 50 µM concentration. However, even 15 µM CdCl2 is toxic in the presence of 1 mM L-buthionine sulfoximine (BSO), suggesting the presence of a Cd-inducible phytochelatin and its involvement in Cd-tolerance in cauliflower. To develop heavy metal-tolerant transgenic plants, we ligated the structural gene of yeast metallothionein gene (CUP1) downstream of CaMV35S promoter and introduced the fused gene into cauliflower. A Cd-tolerant transgenic cauliflower was selected, which grew well in the presence of 400 µM or less Cd, whereas the non-transformed cauliflower tolerated only up to 25 µM Cd. The transgenic cauliflower accumulated more Cd, especially in the upper leaves, than the non-transformed plant.In conclusion, by transfer of the yeast metallothionein gene into cauliflower increased Cd-tolerance and Cd-accumulating ability can be conferred to the plant.     

酸性土壤上植物应对铝胁迫的过程与机制

铝胁迫是酸性土壤上影响作物产量最重要的因素之一.目前,全球土壤酸化程度进一步加剧了铝胁迫.植物可通过将铝离子与有机酸螯合储藏于液泡和从根系中排出铝毒.排出铝毒主要通过苹果酸转运蛋白ALMT和柠檬酸转运蛋白MATE的跨膜运输来实现.编码ABC转运蛋白和锌指转录因子的基因与植物抗铝胁迫有关.这些抗铝毒基因的鉴别使得通过转基因和分子标记辅助育种等生物技术来提高农作物的抗铝毒能力成为可能.最后提出了植物抗铝胁迫研究中需要解决的关键问题及今后的研究方向.     

The extraction by soil and absorption by plants of applied zinc and cadmium

In five consecutive years lettuce, spinach, spring wheat, endive and maize were grown in pots and the effects of native and soil-applied Zn and Cd on plant Zn and Cd concentrations were studied. The normal interactive pattern was antagonistic, Zn reducing plant Cd uptake, and conversely, but less so. Only in loam soil Zn and Cd were synergistic to some extent, plant Zn uptake increasing with applied Cd.When relating total soil Cd/Zn to plant Cd/Zn separate sets of data could be distinguished for loam and sandy soil, each fitting a straight line. The use of 0.1 M CaCl₂ instead of total extractable soil Cd/Zn makes the two sets of data to coalesce around a single straight line. All crops were found to show a positive linear relationship between 0.1 M CaCl₂-extractable soil Cd/Zn and plant Cd/Zn.     

Raman study of in vivo synthesized Zn(II)-metallothionein complexes: structural insight into metal clusters and protein folding

Metallothioneins (MTs) are metal-chelating peptides that play an active role in zinc homeostasis. The participation of metal ligands other than cysteines and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in nonvertebrate MTs. Here, four Zn(II) complexes of invertebrate MTs (mollusc, insect, nematode, and echinoderm) and the Zn(II)-MT complex of the mammalian MT1 isoform, heterologously synthesized in E. coli, were studied by analytic and spectroscopic techniques. By Raman and circular dichroism spectroscopy, new structural informations were obtained. The five analyzed MT isoforms consist largely of beta-turns with the near exclusion of alpha-helical segments. Raman spectroscopy was revealed as an useful tool, providing information about the state of the cysteine sulfur atoms (metal coordinated and oxidized), the participation of histidine in metal coordination, and the molecular environment of tyrosine residues. In all the five Zn(II)-MT studied samples, acid-labile sulfide anions were found as nonproteic ligands, since sulfide-containing and sulfide-devoid species coexisted in the corresponding preparations. Significantly, Raman bands useful as markers of sulfide bridging ligands were identified. Overall, this work illustrates how the combination of analytical and spectroscopic techniques can be a very informative approach for the analysis of in vivo-synthesized metal-MT complexes, providing new data on the metal binding behavior of MTs from the most diverse organisms.     

Concentrations of cadmium,zinc, copper,iron, and metallothionein in liver and kidney of nonhuman primates

To evaluate the species specificity of Cd accumulation and the relationship of Cd with other essential metals and metallothionein (MT), the concentrations of Cd, Zn, Cu, and Fe in the liver and kidney and the MT concentrations in the soluble fractions of the liver and kidney were determined in Cd-uncontaminated nonhuman primates (11 species, 26 individuals) kept in a zoo and two wild-caught Japanese macaques. The compositions of metal-binding proteins in the soluble fractions were also investigated by high-performance liquid chromatography (HPLC). The hepatic Cd concentration was 0.03–14.0 μg/g and the renal Cd concentration was 0.35–99.0 μg/g, both varying greatly and being higher in nonhuman primates, which were more closely related to man. The hepatic Zn concentration was 24.0–176 μg/g and the renal Zn concentration was 13.5–138 μg/g, showing 7- to 10-fold differences, and a correlation (r=0.558, p<0.01) was found between renal Zn and renal Cd concentrations. It was proved that in the liver, MT is more closely correlated with Zn (r=0.795, p<0.001) than with Cd (r=0.492, p<0.01) and that in the kidney MT is correlated with both Cd (r=0.784, p<0.001) and Zn (r=0.742, p<0.001). HPLC analysis of metals bound to MT-like protein in chimpanzees, de Brazza’s monkeys, and Bolivian squirrel monkeys showed that more than 90% of Cd in both the liver and kidney, approx 40% of Zn in liver and 28–69% of Zn in kidney were bound to MT-like protein. The higher percentage Zn was bound to high-molecular protein.     

Increased urinary excretion of zinc and copper by mercuric chloride injection in rats

The effects of HgCl₂ on urinary excretion of Zn, Cu and metallothionein at different time intervals were observed in male Wistar rats. The rats were given a daily intraperitoneal injection of²⁰³HgCl₂ (0.5 or 1.0 mg Hg kg^–1) for 2 days.²⁰³Hg, Zn, Cu and metallothionein in urine, kidney and liver were analyzed. Significant increases in urinary Zn and Cu concentrations were found in HgCl₂-dosed groups. Elevated urinary Zn and Cu concentrations were accompanied by an increased metallothionein excretion in urine at different time periods. Zn concentration in urine remained elevated during the entire observation period of 7 days. There were also increased concentrations of Cu and Zn in the renal cortex in one of the two exposed groups. The results indicate that urinary Cu and Zn are related to the manifestation of renal toxicity and/or the synthesis of metallothionein in kidney induced by mercury.     

The influence of dietary and waterborne zinc on heat-stable metal ligands in rainbow trout, Salmo gairdneri Richardson: quantification by 109Cd radioassay and evaluation of the assay

The ¹⁰⁹Cd binding assay of Eaton & Toal was critically evaluated and then used to assess the induction of cytosolic metal-binding ligands in rainbow trout exposed to Zn in the diet and/or in the water for 16 weeks. With purified rabbit Cd-Zn metallothionein (MT), ¹⁰⁹Cd binding and total Cd recovery in the assay were linear up to 5 μg of protein; gel chromatography revealed a single peak. With heat-denatured extracts of gill, liver and intestine from control and Cd- and Zn-injected trout, ¹⁰⁹Cd binding was generally linear with sample size. Gel chromatography demonstrated that ¹⁰⁹Cd was bound by a protein with the same apparent weight as MT (∼ 11000 daltons), but significant binding occurred also at three other regions [molecular weight (mol wt) >70 000, 30000 and <3000]. In the dietary/waterborne Zn exposure, induced ¹⁰⁹Cd-binding activity occurred not in the MT peak but in the low mol wt peak (< 3000). Activity in the gill rose in response to both dietary and waterborne Zn, but the liver did not respond. The maximum five-fold elevation in the gill was primarily a waterborne effect. In the intestine, the maximum rise was 25-fold due to both factors. The thresholds for induction were > 39 μg Zn^| in water, and > 90 mg kg ^| in the diet, but only when waterborne Zn was also high. There was no correlation between ¹⁰⁹Cd binding and acid soluble thiol levels, which tended to decline at higher Zn exposures.     

四种鳃金龟对不同植物挥发物的触角电位反应

华北大黑鳃金龟Holotrichia oblita Falderman、暗黑鳃金龟Holotrichia parallela Motschulsky、小黄鳃金龟Metabolus flavescens Brenske和福婆鳃金龟Brahmina faldermanni Kraatz有不同的寄主范围.本研究采用溶剂提取法...     

Heavy metal intracellular balance and relationship with metallothionein induction in the liver of carp after contamination by silver,cadmium and mercury following or not pretreatment by zinc

Determination of metal levels (copper, zinc, cadmium, silver and mercury) in soluble and insoluble fractions of liver homogenates has been performed after 7 days exposure of carps (Cyprinus carpio) to moderate concentrations of cadmium, silver and mercury in water. Metallothionein (MT) levels have been quantified by a polarographic method before and after the contamination and a subsequent decontamination phase (7 days). The influence of pretreatment by zinc (7 days) has also been evaluated. MT level variations have been interpreted as having regard to inter-related flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in liver. Regardless of pretreatment, mercury gave the highest increase of liver MT, but the MT level decreased during the decontamination step, especially after pretreatment by zinc. Cadmium and silver gave similar increases, but a significant difference with the control appeared only after the decontamination step with cadmium, while 1 week of contamintion was enough for silver. However, silver binding with MT was achieved only by the end of the decontamination step, while cadmium depicted the highest ratio for HSC-bound toxic metals after the contamination. Our experimental conditions gave the following order of potency for MT induction in liver: mercury silver > cadmium > zinc. Results are discussed comparatively with data obtained with carp gills.     

狮子山铜尾矿植物对铜的吸收及土壤特的影响

我国铜矿储藏丰富,铜矿开采带来巨大经济利益的同时,也对生态环境造成极大的破坏,这种恶劣的环境严重阻碍了植物的定居,但是自然界物种繁多,总有一些植物能适应这种环境而生存下来.本文通过对狮子山优势植物吸收和积累铜的分析,发现这些植物均能富集较多的铜,在土壤铜含量很高的情况下,依然生长旺盛,没有出现受害症状,成为尾矿上的优势种,并形成了单优群落或多优小群落.这些植物的存在改变了土壤的理化特性,降低了土壤中的重金属的含量,提高了土壤的全N、全P、全K和有机质含量,一定程度上改善了土壤的不良环境,在尾矿的植被恢复和土壤修复中起着非常重要的作用.     

Mobilization of iron and other micronutrient cations from a calcareous soil by plant-borne,microbial, and synthetic metal chelators

Mobilization of Fe, Zn, Cu, and Mn by various chelators from a calcareous soil was measured using a simple dialysis tube/complexing resin system. Root exudates from Fe-deficient barley increased the concentrations of all four metals in solution by, on average, a factor of 20, and the addition of complexing resin as a sink for heavy metal cations forced steady state solution concentrations to be reached sooner. Root exudates mobilized increasing amounts of the various micronutrients in the following order: Cu<Fe<Zn<Mn. Phytosiderophores isolated from root exudates of Fe-deficient barley mobilized similar amounts of Cu and Zn but somewhat more Fe and considerably more Mn than crude exudate. The synthetic chelators EDDHA and DTPA showed low specificity in micronutrient mobilization, but the microbial siderophore Desferal was relatively more specific, preferentially mobilizing Fe and Mn. The data indicates that phytosiderophores are capable of increasing the amount of complexed cations in solution. Despite their lack of specificity, phytosiderophores were just as effective as Desferal increasing the availability of Fe. Thus, phytosiderophores, as plant-borne chelators, are certainly of significance for the Fe nutrition of cereals grown in calcareous soils.     

Heavy metal intracellular balance and relationship with metallothionein induction in the gills of carp

Determination of metal levels (Cu, Zn, Cd, Ag, Hg) in soluble and insoluble fractions of gill homogenates has been performed after 7 d exposure of carp (Cyprinus carpio) to moderate concentrations of Cd, Ag, and Hg in water. Metallothionein levels have been quantified by polarographic method before and after contamination and a subsequent decontamination phase (7 d). The influence of pretreatment by zinc (7 d) has also been evaluated. Metallothionein level variations have been interpreted as having regard to interrelated flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in gill. Regardless of pretreatment, mercury gave the highest increase of gill MT, and after the decontamination MT level remained high compared to control. Cadmium and silver gave similar increases, but a significant difference with control appeared only after the decontamination step with silver, whereas 1 week of contamination was enough for cadmium. Our experimental conditions gave the following order of potency for MT induction in gill: Hg≫Cd>Ag>Zn.     

Mobilisation of iron and manganese by plant-borne and synthetic metal chelators

Phytosiderophores released by roots of iron-deficient grasses mobilise Fe, Zn, Mn and Cu in calcareous soils. Mobilisation of Fe, Zn and Cu can be explained as the chelation of these metal cations by phytosiderophores. Mobilisation of Mn could not be so explained because phytosiderophores have a much smaller affinity for Mn than for Fe, Cu and Zn. Model experiments have been made with freshly precipitated Fe(OH)₃ and different soils to study the mobilisation of iron and manganese by plant-borne chelating phytosiderophores, the synthetic metal chelators DTPA and the microbial metal chelator sulphonated ferrioxamine B (FOB). Compared with the synthetic chelator DTPA, the plant-borne chelating phytosiderophores mobilised Fe very efficiently, but no change was observed in the Mn mobilisation by phytosiderophores.Different phytosiderophores, as well as the microbial metal chelator FOB, were used to compare the mobilisation of iron and manganese in a calcareous soil.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.     

Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors

The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri. We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT-PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (CapLC-ESI-QTOF-MS)-based NA analysis procedure and consistently found higher NA levels in roots of A. halleri. Expression of a NA synthase in Zn2+-hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri. Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species.     

一氧化氮在植物体内的来源和功能

一氧化氮(nitric oxide,NO)是生物体内重要的活性分子。NO参与了动物体内血管松弛、神经传递及免疫防御反应等一系列生理功能而被认为是可扩散的多功能第二信使。在植物体内NO也是一种广泛存在的信号分子,参与调节了许多重要的生理过程如生长、发育、抗病防御反应、细胞程序性死亡和抗逆反应。对NO在植物体内的来源、信号转导、调节植物生长发育和对胁迫的响应方面所发挥的作用进行了综述,并讨论了其潜在的一些功能。     

The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress

The effect of heavy metal stress as a potent abiotic elicitor for triggering an accumulation of jasmonic acid (JA) was investigated. Copper and cadmium in in vivo conditions induced accumulation of jasmonates in mature leaves of Arabidopsis thaliana and in young and oldest Phaseolus coccineus plants. The dynamics of jasmonate accumulation showed a biphasic character in both plants. In the first phase, after 7 (A. thaliana) or 14 h (P. coccineus) of exposure to Cu or Cd, a rapid increase of JA level occurred, followed by a rapid decrease observed during 7 successive hours. In the next phase, a repeated but slow increase of JA content occurred. The heavy metal stress induced in particular a more stable (3R,7R) form of jasmonates. These results indicate that JA is connected with the mechanism of toxic action of both heavy metals in plants, differentially reacting to exogenous JA and possessing variable dynamics depending on the plants studied as well as their growth stage.