Anna Ruszczyńska

Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation

Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications.

The role of subcellular distribution of cadmium and phytochelatins in the generation of distinct phenotypes of AtPCS1- and CePCS3-expressing tobacco

Exposure to Cd2+ leads to activation of phytochelatin synthase (PCS) and the formation of phytochelatins (PCs) in the cytosol. Binding of Cd by PCs and the subsequent transport of PC-Cd complexes to the vacuole are essential for Cd tolerance. Attempts to improve Cd detoxification by PCS overexpression have resulted in contrasting plant phenotypes, ranging from enhanced Cd tolerance to Cd hypersensitivity. In the present paper, changes in the subcellular phytochelatin, glutathione, gamma-glutamylcysteine and cadmium vacuolar and cytosolic distribution underlying these phenotypes were examined. Cadmium and PCs levels were determined in protoplasts and vacuoles isolated from leaves of Nicotiana tabacum expressing either of two phytochelatin synthase genes, AtPCS1 and CePCS (differing in their level of Cd tolerance; being Cd hypersensitive or more Cd-tolerant as compared to wild-type plants, respectively). We showed that Cd hypersensitivity of AtPCS1-expressing tobacco results from a significant decrease in both the cytosolic and vacuolar pool of PCs, indicating a decreased cadmium detoxification capacity. By contrast, enhanced Cd tolerance of CePCS plants was accompanied by an increased cytosolic and vacuolar SH of PC/Cd ratio, suggesting more efficient Cd detoxification. Surprisingly, the substantially reduced level of PCs did not influence Cd accumulation in vacuoles of AtPCS1-transformed tobacco (relative to the wild-type), which suggests the important role of mechanisms other than PC-Cd transport in Cd translocation to the vacuole. Our data suggest that the key role of the PCs in Cd tolerance is temporary binding of Cd2+ in the cytosol, and contrary to the current view, their contribution to cadmium sequestration seems to be less important.

HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier

Ectopic expression in tobacco (Nicotiana tabacum v. Xanthi) of the export protein AtHMA4 (responsible in Arabidopsis for the control of Zn/Cd root to shoot translocation) resulted in decreased Cd uptake/accumulation in roots and shoots. This study contributes to understanding the mechanisms underlying this Cd-dependent phenotype to help predict the consequences of transgene expression for potential phytoremediation/biofortification-based strategies. Microarray analysis was performed to identify metal homeostasis genes that were differentially expressed in roots of Cd-exposed AtHMA4-expressing tobacco relative to the wild type. It was established that down-regulation of genes known to mediate Cd uptake was not responsible for reduced Cd uptake/accumulation in AtHMA4 transformants. The transcript levels of NtIRT1 and NtZIP1 were higher in transgenic plants, indicating an induction of the Fe and Zn deficiency status due to AtHMA4 expression. Interestingly, upon exposure to Cd, genes involved in cell wall lignification (NtHCT, NtOMET, and NtPrx11a) were up-regulated in transformants. Microscopic analysis of roots demonstrated that expression of AtHMA4 caused an induction of cell wall lignification in the external cell layers that was accompanied by enhanced H2O2 accumulation. Further study showed that the concentration of other elements (B, Co, Cu, Ni, Mo, and Zn) was reduced in AtHMA4 transformants in the presence of Cd. In conclusion, due to ectopic expression of 35S::AtHMA4, the physical apoplastic barrier within the external cell layer developed, which is likely to be responsible for the reduction of Cd uptake/accumulation.

Metal response of transgenic tomato plantsexpressing P1B-ATPase

Heterologous expression of HMA4 (P(1B) -ATPase) in plants is a useful strategy to engineer altered metal distribution in tissues for biofortification or phytoremediation purposes. This study contributes to understanding mechanisms underlying complex Zn-dependent phenotypes observed in transgenic plants and to better predict the consequences of transgene expression. Tomato was transformed with AhHMA4(p1) ::AhHMA4 from Arabidopsis halleri encoding the Zn export protein involved in xylem loading of Zn. Homozygous lines were tested for Zn tolerance, Zn and Fe concentrations in organs and in the apoplastic fluid, and for the expression of the transgene and tomato metal homeostasis endogenes. Expression of AhHMA4 facilitates root-to-shoot Zn translocation and induces Zn uptake in a Zn supply-dependent manner. Unexpectedly, it increases Zn excess-triggered Fe deficiency in leaves and transcriptional activation of Fe-uptake systems in roots. Moreover, AhHMA4 expression causes Zn overload of the apoplast, which may contribute to enhanced Zn sensitivity of transgenics and may lead to cell-wall remodeling. This study highlights that alteration of the apoplast/symplast Zn status through introduction of cellular Zn export activity via AhHMA4 may alter tomato metal homeostasis network, thus seems to be crucial in the generation of the phenotype of transgenic tomato.

Expression of HvHMA2 in tobacco modifies Zn-Fe-Cd homeostasis.

HvHMA2 is a plasma membrane P1B-ATPase from barley that functions in Zn/Cd root-to-shoot transport. To assess the usefulness of HvHMA2 for modifying the metal content in aerial plant parts, it was expressed in tobacco under the CaMV35S promoter. Transformation with HvHMA2 did not produce one unique pattern of Zn and Cd accumulation; instead it depended on external metal supply. Thus Zn and Cd root-to-shoot translocation was facilitated, but not at all applied Zn/Cd concentrations. Metal uptake was restricted in HvHMA2-transformed plants and the level in the shoot was not enhanced. It was shown that HvHMA2 localizes to the plasma membrane of tobacco cells, and overloads the apoplast with Zn, which could explain the overall decrease in metal uptake observed. Despite the lower levels in the shoot, HvHMA2 transformants showed increased Zn sensitivity. Moreover, introduction of HvHMA2 into tobacco interfered with Fe metabolism and Fe accumulation was modified in HvHMA2-transformants in a Zn- and Cd-concentration dependent manner. The results indicate that ectopic expression of the export protein HvHMA2 in tobacco interferes with tobacco metal Zn-Cd-Fe cross-homeostasis, inducing internal mechanisms regulating metal uptake and tolerance.

The ratio of Zn to Cd supply as a determinant of metal-homeostasis gene expression in tobacco and its modulation by overexpressing the metal exporter AtHMA4

Highlight Modifications of endogenous metal-homeostasis traits, in particular co-ordinated regulation of NtZIP1, NtZIP4, NtIRT1-like, and NtVTL, contribute to the generation of the phenotype of AtHMA4-expressing tobacco.

Highly efficient and time economical purification of olefin metathesis products from metal residues using an isocyanide scavenger

A sustainable protocol of olefin metathesis in non-degassed, undistilled ethyl acetate under air with commercially available self-scavenging ruthenium catalysts is described. Furthermore, a time economical, cost-effective and scalable method of removal of ruthenium residues is presented. Treatment of post-reaction mixtures with an isocyanide scavenger and then with acid followed by a simple filtration is shown to yield OM products with ruthenium contamination below 5 ppm even in highly challenging cases. Finally, a telescope RCM/Suzuki–Miyaura sequence with a rapid and efficient purification protocol for simultaneous removal of Ru and Pd residues from solution is described.

Seleno-compounds and Carnosic Acid Added to Diets with Rapeseed and Fish Oils Affect Concentrations of Selected Elements and Chemical Composition in the Liver, Heart and Muscles of Lambs

The objective of our studies was to investigate effects of carnosic acid (CA), selenized yeast (SeY) and selenate (SeVI) added to the diet including rapeseed oil (RO) and fish oil (FO) on concentrations of elements, fatty acids (FAs), tocopherols, cholesterol, and malondialdehyde in the liver, heart, musculus longissimus dorsi (MLD), and musculus biceps femoris (MBF) of lambs. Lambs were fed diets: group I—the basal diet (BD) with RO; group II—BD with RO and FO; group III—BD with RO, FO, and CA; group IV—BD with RO, FO, CA, and SeY; group V—BD with RO, FO, CA, and SeVI. The diets with Se compounds increased Se concentrations in all tissues compared with other diets. The diet with SeVI increased Cd, Sb, and Pb concentrations in the liver compared to groups I, II, and IV. The diets containing Se compounds increased Sb and Pb concentrations in MBF compared to groups I and II. All diets with CA reduced As, Sb, and Pb concentrations in MLD compared to groups I and II. All diets with FO increased concentrations of FAs and malondialdehyde in the liver compared to group I. All diets with FO decreased FAs concentrations in MBF compared to group I. The diets containing CA with/without Se compounds increased malondialdehyde concentrations in MBF compared to groups I and II. The diet with Se compounds reduced malondialdehyde concentrations in MLD compared to group II. All diets with FO changed concentrations of tocopherols and cholesterol in all tissues compared to group I. Our study showed that the addition of SeY or SeVI to the experimental diet increased the concentration of Se in all assayed tissues of lambs without adversely influencing performance or causing physiological disorders in internal organs. Both, SeY or SeVI added to the experimental diet decreased the oxidative stress and the concentrations of As, Sb, and Pb in MLD compared with the diets containing RO, irrespective of the presence of FO (groups I and II). Our study provides useful knowledge for nutritionists carrying out further investigations aimed at improving farm animal health, performance, and the nutritional quality of animal products for humans.

Analytical Techniques for Trace Element Determination

A lot of elements occur in different matrices at low levels of content, and a lot of these elements were not detectable by analytical methods for a long time. The knowledge about their presence appeared with the development of analytical technology and caused the origin of the term “trace elements.” Trace element defined by IUPAC [1] is any element having an average concentration of less than about 100 parts per million atoms or less than 100 mg/kg. In the second half of the 20th century, together with rapid increase of detection capabilities of analytical techniques, a new term of ultratrace elements appeared. Even though the term exists and is commonly used, there is no rigid definition. Ultratrace concerns elements at mass fraction below 1 ppm. The knowledge of trace and ultratrace elements is very important in various fields of science, industry, and technology. Ultralow concentrations of elements might be as well essential as hazardous doses for organisms; some traces can dramatically change properties of designed devices. Therefore, the need for accurate measurements at low amount of contents is required and very important. The common use of extremely sensitive instrumentation needs the adequate control of contamination and verification of the accuracy of determination. The gain of analytical sensitivity multiplied contamination as well as other problems. Therefore, correct precautions should be taken to determine trace elements in the parts per billion concentration range and below. Errors during trace and ultratrace elemental analysis can be caused by improper sampling, storage, sample preparation, and, finally, by analysis itself. Therefore, an accuracy of an analytical determination should be always established. Collecting a representative sample without contaminating is a key to the meaningful analysis and Thiers’ words from 1957 “unless the complete history of any given sample is known with certainty, the analyst is well advised not to spend his time analyzing it” [2] is always up to date. Nowadays, there are a large number of available analytical techniques allowing for trace and ultratrace analysis of elemental composition. For the trace elements that are present in parts per million concentration range, the most widely used technique is probably atomic absorption spectrometry with flame atomization. For ultratrace elements present in concentration of parts per billion and below, the number of suitable techniques drops due to the required analytical sensitivity. The determination of trace elements is commonly held with potentiometry, voltammetry, atomic spectrometry, X-ray, and nuclear methods. Electrochemical methods can measure either free ions in solution (potentiometry) or free ions together with ions bound in labile complexes (voltammetry), and they can also provide analysis of the oxidation state of some of the elements. Atomic spectrometric techniques are very sensitive and can be used to measure the total element content within a sample; however, accuracy of these techniques can be affected by the matrix of the sample. X-ray and nuclear techniques provide very low limit of detections and matrix insensitivity and are used for comparison of results due to their principles fundamentally different from those of the other analytical techniques. Therefore, they are less likely to be prone to the same systematic biases. Benefits and losses of each technique should concern the number of analytes possible to measure with the use of the technique, occurrence of interferences and difficulties, detection limits, throughput of samples, and expenses. The determination of trace elements and contaminants in complex matrices often requires extensive sample preparation and/or extraction prior to instrumental analysis. A large number of samples that need to have determined the concentration of essential and toxic elements belong to food [3, 4], environmental [5, 6], clinical and biological [5–7, 7–9] samples. Routinely, the determination of trace metals has been carried out by inductively coupled plasma atomic emission spectrometry (ICPAES), inductively coupled plasma mass spectrometry (ICPMS), electrothermal atomic absorption spectrometry (ETAAS), and flame atomic absorption spectrometry (FAAS). However, matrix of many samples (biological, clinical, environmental, etc.) is complex and consists of high amounts of soluble solid substances and large amounts of inorganic compounds (i.e., salts of Ca, K,

Determination the Usefulness of AhHMA4p1::AhHMA4 Expression in Biofortification Strategies

AhHMA4 from Arabidopsis thaliana encodes Zn/Cd export protein that controls Zn/Cd translocation to shoots. The focus of this manuscript is the evaluation of AhHMA4 expression in tomato for mineral biofortification (more Zn and less Cd in shoots and fruits). Hydroponic and soil-based experiments were performed. Transgenic and wild-type plants were grown on two dilution levels of Knop’s medium (1/10, 1/2) with or without Cd, to determine if mineral composition affects the pattern of root/shoot partitioning of both metals due to AhHMA4 expression. Facilitation of Zn translocation to shoots of 19-day-old transgenic tomato was noted only when plants were grown in the more diluted medium. Moreover, the expression pattern of Zn-Cd-Fe cross-homeostasis genes (LeIRT1, LeChln, LeNRAMP1) was changed in transgenics in a medium composition-dependent fashion. In plants grown in soil (with/without Cd) up to maturity, expression of AhHMA4 resulted in more efficient translocation of Zn to shoots and restriction of Cd. These results indicate the usefulness of AhHMA4 expression to improve the growth of tomato on low-Zn soil, also contaminated with Cd.