The role of the kinase OXI1 in cadmium‐ and copper‐induced molecular responses in Arabidopsis thaliana

The hypothesis that mitogen‐activated protein kinase (MAPK) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of oxidative signal‐inducible kinase (OXI1) in metal‐induced oxidative signalling, the responses of oxi1 knockout lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared with those of wild‐type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase‐1 may be a downstream component of OXI1 signalling. Metal‐specific differences in OXI1 action were observed. For example, OXI1 was required for the up‐regulation of antioxidative defences such as catalase in leaves and Fe‐superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1‐MKK2‐WRKY25 cascade. Moreover, the induction of Cu/Zn superoxide dismutases in Cu‐exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1‐independent pathways but rather involves changes in components mediating intracellular communication.     

Overexpression of membrane-associated acyl-CoA-binding protein ACBP1 enhances lead tolerance in Arabidopsis

In Arabidopsis thaliana , a family of six genes encodes acyl-CoA-binding proteins (ACBPs) that show conservation at the acyl-CoA-binding domain. They are the membrane-associated ACBP1 and ACBP2, extracellularly targeted ACBP3, kelch-motif-containing ACBP4 and ACBP5, and 10-kDa ACBP6. The acyl-CoA domain in each of ACBP1 to ACBP6 binds long-chain acyl-CoA esters in vitro , suggestive of possible roles in plant lipid metabolism. We addressed here the use of Arabidopsis ACBPs in conferring lead [Pb(II)] tolerance in transgenic plants because the 10-kDa human ACBP has been identified as a molecular target for Pb(II) in vivo . We investigated the effect of Pb(II) stress on the expression of genes encoding Arabidopsis ACBP1, ACBP2 and ACBP6. We showed that the expression of ACBP1 and ACBP2 , but not ACBP6 , in root is induced by Pb(II) nitrate treatment. In vitro Pb(II)-binding assays indicated that ACBP1 binds Pb(II) comparatively better, and ACBP1 was therefore selected for further investigations. When grown on Pb(II)-containing medium, transgenic Arabidopsis lines overexpressing ACBP1 were more tolerant to Pb(II)-induced stress than the wild type. Accumulation of Pb(II) in shoots of the ACBP1 -overepxressing plants was significantly higher than wild type. The acbp1 mutant showed enhanced sensitivity to Pb(II) when germinated and grown in the presence of Pb(II) nitrate and tolerance was restored upon complementation using an ACBP1 cDNA. Our results suggest that ACBP1 is involved in mediating Pb(II) tolerance in Arabidopsis with accumulation of Pb(II) in shoots. Such observations of Pb(II) accumulation, rather than Pb(II) extrusion, in the ACBP1 -overexpressing plants implicate possible use of ACBP1 in Pb(II) phytoremediation.     

A switch‐off fluorescence probe towards Pb(II) and cu(II) ions based on a calix[4]pyrrole bearing amino‐quinoline group

A new fluorescence receptor calix[4]pyrrole‐N‐(quinoline‐8‐yl) acetamide (CAMQ) containing a pyrrolic ring connected via the meso‐position was synthesized, purified and characterized by elemental analysis, NMR and mass spectroscopy. This compound was examined for its fluorescence properties towards different metal ions e.g. Ag(I), Hg(II), Co(II), Ca(II), Ni(II), Zn(II), Cr(II), Ba(II), Fe(II), Cu(II), Pb(II)and Mg(II) ions by spectrophotometry and spectrofluorometry. It was concluded that the compound (CAMQ) possessed significantly enhanced selectivity for Pb(II) and Cu(II) ions in dimethyl sulfoxide (DMSO) even at very low concentrations (1 μM). It exhibit ‘turn‐on’ fluorescence when exposed to Pb(II) and Cu(II) and did so in preference to other metal ions. The binding constants, stoichiometry and quantum yields have been determined. The quenching mechanism was assessed using the Stern–Volmer equation and was also discussed.     

Arabidopsis acyl‐CoA‐binding protein ACBP1 participates in the regulation of seed germination and seedling development

A family of six genes encoding acyl‐CoA‐binding proteins (ACBPs), ACBP1–ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1‐over‐expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over‐production in 12‐day‐old seedlings up‐regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress‐responsive genes: ABA‐RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH‐TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12‐day‐old seedlings of ACBP1‐over‐expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two‐hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA‐mediated seed germination and seedling development.     

真菌吸附铅锌的研究

正随着采矿业的迅速发展,越来越多的重金属通过多种途径进入土壤环境中,对生态环境造成了不可估量的破坏并严重威胁人类健康。铅锌在工业上具有非常重要的作用且其应用极为广泛,而他们具有的难去除、难迁移和生物累积等特性使得铅锌在环境中的污染尤为突出。通过微生物的生长代谢,有效降低土壤重金属毒性,是促进植物生长的重要步骤之一。同时也要求微生物自身具有抵抗重金属的功能,根际微生     

耐性真菌HA吸附铅、锌的影响因素及吸附机理研

【目的】从铅锌矿区分离筛选出耐铅锌性菌株,并研究其吸附铅、锌的影响因素及吸附机理,为重金属污染微生物修复提供参考。【方法】以从铅锌矿区筛选出的耐铅锌真菌HA作为试验菌株,考察影响其对铅、锌去除能力的主要因素(初始浓度、pH、接种量),同时通过等温吸附模型、动力学分析及红外光谱分析探讨其相关的吸附机理。【结果】经鉴定,从矿区筛选的对Pb2+、Zn2+有较强耐性的菌株HA为米曲霉(Aspergillus oryzae);在初始铅、锌浓度的实验范围内(100-800mg/L),HA对铅、锌离子的去除率随着其初始浓度增加而减小,25h后HA的生长进入平稳期,且对铅、锌离子的去除率趋于稳定。当铅、锌初始浓度为100mg/L、pH为5.0时,HA对铅、锌离子的去除率均达到最高,分别为97.8%、54.1%;当HA接种量为1mL时,其对铅、锌去除率的增长率达到最大。HA对铅、锌的吸附过程满足Langmuir吸附模型,其吸附以单层吸附为主。在动态吸附过程中,HA对Pb2+、Zn2+离子吸附性能与准二级动力学吸附方程的拟合程度更高,且对Pb2+的吸附效果明显高于Zn2+。红外光谱分析表明,HA细胞中羟基、烷基、酰胺基、羰基、磷酸基等参与了Pb2+、Zn2+的吸附过程。【结论】HA是一株对铅、锌有较强吸附能力的真菌,其吸附铅、锌影响因素及吸附机理研究结果将为重金属污染微生物修复提供指导。     

Acyl‐CoA‐binding protein 2 binds lysophospholipase 2 and lysoPC to promote tolerance to cadmium‐induced oxidative stress in transgenic Arabidopsis

Lysophospholipids are intermediates of phospholipid metabolism resulting from stress and lysophospholipases detoxify lysophosphatidylcholine (lysoPC). Many lysophospholipases have been characterized in mammals and bacteria, but few have been reported from plants. Arabidopsis thaliana lysophospholipase 2 (lysoPL2) (At1g52760) was identified as a protein interactor of acyl‐CoA‐binding protein 2 (ACBP2) in yeast two‐hybrid analysis and co‐immunoprecipitation assays. BLASTP analysis indicated that lysoPL2 showed ～35% amino acid identity to the lysoPL1 family. Co‐localization of autofluorescence‐tagged lysoPL2 and ACBP2 by confocal microscopy in agroinfiltrated tobacco suggests the plasma membrane as a site for their subcellular interaction. LysoPL2 mRNA was induced by zinc (Zn) and hydrogen peroxide (H₂O₂), and lysoPL2 knockout mutants showed enhanced sensitivity to Zn and H₂O₂ in comparison to wild type. LysoPL2‐overexpressing Arabidopsis was more tolerant to H₂O₂ and cadmium (Cd) than wild type, suggesting involvement of lysoPL2 in phospholipid repair following lipid peroxidation arising from metal‐induced stress. Lipid hydroperoxide (LOOH) contents in ACBP2‐overexpressors and lysoPL2‐overexpressors after Cd‐treatment were lower than wild type, indicating that ACBP2 and lysoPL2 confer protection during oxidative stress. A role for lysoPL2 in lysoPC detoxification was demonstrated when recombinant lysoPL2 was observed to degrade lysoPC in vitro. Filter‐binding assays and Lipidex competition assays showed that (His)₆‐ACBP2 binds lysoPC in vitro. Binding was disrupted in a (His)₆‐ACBP2 derivative lacking the acyl‐CoA‐binding domain, confirming that this domain confers lysoPC binding. These results suggest that ACBP2 can bind both lysoPC and lysoPL2 to promote the degradation of lysoPC in response to Cd‐induced oxidative stress.     

A novel fluorescent sensor based on electrosynthesized benzene sulfonic acid‐doped polypyrrole for determination of Pb(II) and Cu(II)

To develop conducting organic polymers (COPs) as luminescent sensors for determination of toxic heavy metals, a new benzene sulfonic acid‐doped polypyrrole (PPy‐BSA) thin film was electrochemically prepared by cyclic voltammetry (CV) on flexible indium tin oxide (ITO) electrode in aqueous solution. PPy‐BSA film was characterized by FTIR spectrometry, X‐ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The optical properties of PPy‐BSA were investigated by ultraviolet (UV)‐visible absorption and fluorescence spectrometry in dimethylsulfoxide (DMSO) diluted solutions. PPy‐BSA fluorescence spectra were strongly quenched upon increasing copper(II) ion (Cu²⁺) and lead(II) ion (Pb²⁺) concentrations in aqueous medium, and linear Stern–Volmer relationships were obtained, which indicated the existence of a main dynamic fluorescence quenching mechanism. BSA‐PPy sensor showed a high sensitivity for detection of both metallic ions, Cu²⁺ and Pb²⁺, with very low limit of detection values of 3.1 and 18.0 nM, respectively. The proposed quenching‐fluorimetric sensor might be applied to the determination of traces of toxic heavy metallic ions in water samples.     

Modeling of Pb(II) adsorption by a fixed-bed column

Removal of Pb(II) from an aqueous environment using biosorbents is a cost-effective and environmentally benign method. The biosorption process, however, is little understood for biosorbents prepared from plant materials. In this study, the biosorption process was investigated by evaluating four adsorption models. A fixed-bed column was prepared using a biosorbent prepared from the aquatic plant Hydrilla verticillata. The effect of bed height and flow rate on the biosorption process was investigated. The objective of the study was to determine the ability of H. verticillata to biosorb Pb(II) from an aqueous environment and to understand the process, through modeling, to provide a basis to develop a practical biosorbent column. Experimental breakthrough curves for biosorption of 50 mg L^?1 aqueous Pb(II) using a fixed-bed column with 1.00 cm inner diameter were fitted to the Thomas, Adams-Bohart, Belter, and bed depth service time (BDST) models to investigate the behavior of each model according to the adsorption system and thus understand the adsorption mechanism. Model parameters were evaluated using linear and nonlinear regression methods. The biosorbent removed 65% (82.39 mg g^?1 of biosorbent) of Pb(II) from an aqueous solution of Pb(NO₃)₂ at a flow rate of 5.0 ml min^?1 in a 10 cm column. Na₂CO₃ was used to recover the adsorbed Pb(II) ions as PbCO₃ from the biosorbent. The Pb(II) was completely desorbed at a bed height of 10.0 cm and a flow rate of 5.0 ml min^?1. Fourier transform infrared (FT-IR) analysis of the native biosorbent and Pb(II)-loaded biosorbent indicated that the hydroxyl groups and carboxylic acid groups were involved in the metal bonding process. The FT-IR spectrum of Pb(II)-desorbed biosorbent showed an intermediate peak shift, indicating that Pb(II) ions were replaced by Na⁺ ions through an ion-exchange process. Of the four models tested, the Thomas and BDST models showed good agreement with experimental data. The calculated bed sorption capacity N₀ and rate constant k_a were 31.7 g L^?1 and 13.6 × 10^?4 L mg^?1 min^?1 for the C_t/C₀ value of 0.02. The BDST model can be used to estimate the column parameters to design a large-scale column.     

Characterization of Bacteria in the Rhizosphere Soils of Polygonum Pubescens and Their Potential in Promoting Growth and Cd,Pb, Zn Uptake by Brassica napus

Microbe-enhanced phytoremediation has been considered as a promising measure for the remediation of metal-contaminated soils. In this study, two bacterial strains JYX7 and JYX10 were isolated from rhizosphere soils of Polygonum pubescens grown in metal-polluted soil and identified as of Enterobacter sp. and Klebsiella sp. based on 16S rDNA sequences, respectively. JYX7 and JYX10 showed high Cd, Pb and Zn tolerance and increased water-soluble Cd, Pb and Zn concentrations in culture solution and metal-added soils. Two isolates produced plant growth-promoting substances such as indole acetic acid, siderophore, 1-aminocyclopropane-1-carboxylic deaminase, and solubilized inorganic phosphate. Based upon their ability in metal tolerance and solubilization, two isolates were further studied for their effects on growth and accumulation of Cd, Pb, and Zn in Brassica napus (rape) by pot experiments. Rapes inoculated with JYX7 and JYX10 had significantly higher dry weights, concentrations and uptakes of Cd, Pb, Zn in both above-ground and root tissues than those without inoculation grown in soils amended with Cd (25 mg kg^?1), Pb (200 mg kg^?1) or Zn (200 mg kg^?1). The present results demonstrated that JYX7 and JYX10 are valuable microorganism, which can improve the efficiency of phytoremediation in soils polluted by Cd, Pb, and Zn.     

Complexation of lead(II) by nicotinamide (nia): Crystal structure of polymeric Pb(nia)(NO3)2

Lead(II) nitrate complex with nicotinamide has been synthesized and characterized by elemental analysis, IR, ¹H NMR, ¹³C NMR, UV-Vis spectroscopy and X-ray crystallography. The Pb²⁺ centres linked through nicotinamide and nitrate(V) ligands form a two-dimensional polymeric structure defining a double layer and each layer is also stabilized by hydrogen bonds of N-H?O and C-H?O type.     

Sorption of Cd(II) and Pb(II) by exopolymeric substances (EPS) extracted from activated sludges and pure bacterial strains: Modeling of the metal/ligand ratio effect and role of the mineral fraction

The present study deals with the sorption of Cd(II) and Pb(II) by exopolymeric substances (EPS) extracted from activated sludges or pure bacterial strains. The percentage of sorbed metal increases with the concentration of the EPS–water solution. Pb(II) always presents a higher affinity than Cd(II) for EPS. For the EPS extracted from pure bacterial strains, only one global binding constant from a simple equilibrium sorption model, may be used to assess the effect of microbial products such as EPS on Cd(II) and Pb(II) speciation or mobility in the environment. However, for EPS extracted from activated sludges, the wide variation of the global binding constants determined for Cd(II) and Pb(II) do not permit such a simple approach. The differences in sorption to metals between the two types of EPS (bacterial, activated sludges) could be explained by the differences in EPS composition: organic macromolecules, as well as the nature of the mineral fraction.     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

Imidazolate-bridged dicopper(II) and copper(II)–zinc(II) complexes of macrocyclic ligand with methylimidazol pendants: Model study of copper(II)–zinc(II) superoxide dismutase

Two new homo- and hetero-dinuclear complexes, [Cu₂L(im)](ClO₄)₃4H₂O (1) and [CuZnL(im)](ClO₄)₃4H₂O (2) (where Im=1H-1midazole and L = 3, 6, 9, 16, 19, 22-hexaaza-6, 19-bis(1H-imidazol-4-ylmethyl)tricycle[22, 2, 2, 2^11,14]triaconta-1, 11, 13, 24, 27, 29-hexaene) were synthesized and characterized as model compounds for the active site of copper(II)–zinc(II) superoxide dismutase (Cu₂Zn₂–SOD). X-ray crystal structure analysis revealed that the metal centers in both complexes exhibit distorted trigonal-bipyramid coordination geometry and the CuCu and CuZn distances are both 6.02 Å. Magnetic and ESR spectral measurements of 1 showed antiferromagnetic exchange interactions between the imidazolate-bridged Cu(II) ions. The ESR spectrum of 2 displays typical signals of mononuclear Cu(II) complex, demonstrating the formation of heterodinuclear complex 2 rather than a mixture of homodinuclear Cu(II)/Zn(II) complexes. pH-dependent ESR and UV–visible spectral measurements manifest that the imidazolate exists as a bridging ligand from pH 6 to 11 for both complexes. The IC₅₀ values of 1.96 and 1.57 μM [per Cu(II) ion] for 1 and 2 suggest that they are good models for the Cu₂Zn₂–SOD.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.     

Electrochemical synthesis and crystal structure of iron(II), cobalt(II), nickel(II) and copper(II) complexes of dianionic tetradentate N4 Schiff base ligand

The electrochemical oxidation of anodic metal (iron, cobalt, nickel and copper) in an acetonitrile solution of the potentially chelating Schiff base N,N(dithiodiethylenebis-(aminylydenemethylydene)-bis(1,2-phenylene)ditosylamide (H₂L) afforded stable complexes of empirical formula [ML]. The compounds obtained have been characterized by microanalysis, IR spectroscopy and ES-MS mass spectrometry. The crystal and molecular structures of [FeL]·CH₃CN (1) [CoL]·CH₃CN (2), [NiL]·CH₃CN (3) and [CuL]·CH₃CN (4) have been determined by X-ray diffraction in all complexes, the metal atom is in a distorted tetrahedral environment with the Schiff base acting as a tetradentate N4 donor.