Daniel P. Persson

Iron fortification of rice seeds through activation of the nicotianamine synthase gene

The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression.

Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS

The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as (48)SO(+). Simultaneously, Fe was measured as (72)FeO(+), P as (47)PO(+), and Zn as (66)Zn(+), enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP(6)) was the main Fe binding ligand, with the stoichiometry Fe(4)(IP(6))(18). When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid.

Megapixel imaging of (micro)nutrients in mature barley grains

Understanding the accumulation and distribution of essential nutrients in cereals is of primary importance for improving the nutritional quality of this staple food. While recent studies have improved the understanding of micronutrient loading into the barley grain, a detailed characterization of the distribution of micronutrients within the grain is still lacking. High-definition synchrotron X-ray fluorescence was used to investigate the distribution and association of essential elements in barley grain at the micro scale. Micronutrient distribution within the scutellum and the embryo was shown to be highly variable between elements in relation to various morphological features. In the rest of the grain, the distribution of some elements such as Cu and Zn was not limited to the aleurone layer but extended into the endosperm. This pattern of distribution was less marked in the case of Fe and, in particular, Mn. A significant difference in element distribution was also found between the ventral and dorsal part of the grains. The correlation between the elements was not consistent between and within tissues, indicating that the transport and storage of elements is highly regulated. The complexity of the spatial distribution and associations has important implications for improving the nutritional content of cereal crops such as barley.

Micro-scaled high-throughput digestion of plant tissue samples for multi-elemental analysis

BackgroundQuantitative multi-elemental analysis by inductively coupled plasma (ICP) spectrometry depends on a complete digestion of solid samples. However, fast and thorough sample digestion is a challenging analytical task which constitutes a bottleneck in modern multi-elemental analysis. Additional obstacles may be that sample quantities are limited and elemental concentrations low. In such cases, digestion in small volumes with minimum dilution and contamination is required in order to obtain high accuracy data.ResultsWe have developed a micro-scaled microwave digestion procedure and optimized it for accurate elemental profiling of plant materials (1-20 mg dry weight). A commercially available 64-position rotor with 5 ml disposable glass vials, originally designed for microwave-based parallel organic synthesis, was used as a platform for the digestion. The novel micro-scaled method was successfully validated by the use of various certified reference materials (CRM) with matrices rich in starch, lipid or protein. When the micro-scaled digestion procedure was applied on single rice grains or small batches of Arabidopsis seeds (1 mg, corresponding to approximately 50 seeds), the obtained elemental profiles closely matched those obtained by conventional analysis using digestion in large volume vessels. Accumulated elemental contents derived from separate analyses of rice grain fractions (aleurone, embryo and endosperm) closely matched the total content obtained by analysis of the whole rice grain.ConclusionA high-throughput micro-scaled method has been developed which enables digestion of small quantities of plant samples for subsequent elemental profiling by ICP-spectrometry. The method constitutes a valuable tool for screening of mutants and transformants. In addition, the method facilitates studies of the distribution of essential trace elements between and within plant organs which is relevant for, e.g., breeding programmes aiming at improvement of the micronutrient density in edible plant parts. Compared to existing vial-in-vial systems, the new method developed here represents a significant methodological advancement in terms of higher capacity, reduced labour consumption, lower material costs, less contamination and, as a consequence, improved analytical accuracy following micro-scaled digestion of plant samples.

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn2+ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn2+ and Cd2+ chelator with capacity to bind 10 Zn2+ ions per C terminus. When AtHMA4 is expressed in a Zn2+-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn2+ and Cd2+ tolerance and lowered Zn2+ and Cd2+ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn2+ and Cd2+ chelator (sensor) and as a regulator of the efficiency of Zn2+ and Cd2+ export. The identification of a post-translational handle on Zn2+ and Cd2+ transport efficiency opens new perspectives for regulation of Zn2+ nutrition and tolerance in eukaryotes.

Review: The role of atomic spectrometry in plant science

Inductively coupled plasma-mass spectrometry (ICP-MS) is the state-of-the-art technique for multi-elemental analysis of plant tissue. It provides a powerful tool in functional genomics, linking altered elemental profiles of mutants with gene expression and function. In addition, with its unmatched sensitivity, ICP-MS enables characterization of the substrate specificity and regulation of membrane transport proteins. Digestion of plant tissue has traditionally represented a bottleneck due to the low capacity of commercially available equipment. However, recent developments in micro-scaled digestion, combined with semi-quantitative analysis and chemometrics, have enabled high-throughput multi-elemental profiling and multivariate classification of large sample sets, thereby supporting a range of new applications in molecular breeding, quality assessment and authenticity testing of plants. Novel hyphenated techniques based on liquid chromatography and ICP-MS (LC-ICP-MS) have significantly improved the understanding of elemental species and their importance for e.g. the bioactivity of metals in plants. Development of procedures for sample pre-treatment, extraction and multi-dimensional separation now allows characterization of important metallo-biomolecules in plants, such as the coordination complexes of phytochelatins, metallothioneins, nicotianamine and inositol phosphates. These are key ligands involved in ion homeostasis, translocation and long-term storage of elements. Much emphasis has also been given to studies of covalently bound Se and As species, primarily due to their impact on human health. LC-ICP-MS has extensively been complemented by molecular mass spectrometry for structural information of biologically relevant species. This review covers the most recent developments in multi-elemental analysis (Part A) and speciation analysis (Part B) in plant science. A number of relevant cases are presented in order to demonstrate how the analytical developments have unravelled the functional roles of elements in plants science. These cases show that ICP-MS is an essential technology in plant metallomic platforms.

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state-of-the-art quantitative 2D liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size-exclusion chromatography/inductively coupled plasma mass spectrometer (SEC-ICP-MS) method, in order to identify the Zn-binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S-transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn-nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn-nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type-specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.

Multi-elemental speciation analysis of barley genotypes differing in tolerance to cadmium toxicity using SEC-ICP-MS and ESI-TOF-MS

Plants respond to Cd exposure by synthesizing heavy-metal-binding oligopeptides, called phytochelatins (PCs). These peptides reduce the activity of Cd2+ ions in the plant tissues by forming Cd chelates. The main objective of the present work was to develop an analytical technique, which allowed identification of the most prominent Cd species in plant tissue by SEC-ICP-MS and ESI-TOF-MS. An integrated part of the method development was to test the hypothesis that differential Cd tolerance between two barley genotypes was linked to differences in Cd speciation. Only one fraction of Cd species, ranging from 700–1800 Da, was detected in the shoots of both genotypes. In the roots, two additional fractions ranging from 2900–4600 and 6700–15 000 Da were found. The Cd-rich SEC fractions were heart-cut, de-salted and de-metallized using reversed-phase chromatography (RPC), followed by ESI-MS-TOF to identify the ligands. Three different families of PCs, viz. (γGlu-Cys)n-Gly (PCn), (γGlu-Cys)n-Ser (iso-PCn) and Cys-(γGlu-Cys)n-Gly (des-γGlu-PCn), the last lacking the N-terminal amino acid, were identified. The PCs induced by Cd toxicity also bound several essential trace elements in plants, including Zn, Cu, and Ni, whereas no Mn species were detected. Zn, Cu and Ni-species were distributed between the 700–1800 Da and 6700–15 000 Da fractions, whereas only Cd species were found in the 2900–4600 Da fraction dominated by PC3 ligands. Although the total tissue concentration of Cd was similar for the two species, the tolerant barley genotype synthesized significantly more CdPC3 species with a high Cd specificity than the intolerant genotype, clearly indicating a correlation between Cd tolerance and the Cd–PC speciation.

Multi-elemental fingerprinting of plant tissue by semi-quantitative ICP-MS and chemometrics

The multi-elemental capacity of Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is rarely fully utilized in traditional full-quantitative analysis. The main obstacles are limited availability of multi-elemental standards and the need for time-consuming external calibrations. In this study, a novel semi-quantitative quadrupole ICP-MS based method for multi-elemental fingerprinting of plant tissue was developed as a high-throughput alternative to full-quantitative analysis. The main analytical objectives were low data acquisition time ( 70%. In conjunction with chemometrics, the discrimination power of the semi-quantitative results was better than that of full-quantitative analysis. The superior discrimination power of semi-quantitative analysis was maintained, even when it was combined with a high-throughput digestion procedure, which represented a 5 fold reduction in analytical labour consumption. Thus, the large amount of elemental information obtained using semi-quantitative ICP-MS fully outweighed the lack of accuracy compared to full-quantitative analysis. For the first time it is demonstrated that semi-quantitative ICP-MS in combination with chemometrics provides a fast and powerful alternative to traditional full-quantitative ICP-MS. The method developed here constitutes a promising novel analytical tool, which has the potential to mature into a routine procedure for testing e.g. the authenticity and adulteration of food products.

Contrasting effects of nicotianamine synthase knockdown on zinc and nickel tolerance and accumulation in the zinc/cadmium hyperaccumulator Arabidopsis halleri.

Elevated nicotianamine synthesis in roots of Arabidopsis halleri has been established as a zinc (Zn) hyperaccumulation factor. The main objective of this study was to elucidate the mechanism of nicotianamine-dependent root-to-shoot translocation of metals. Metal tolerance and accumulation in wild-type (WT) and AhNAS2-RNA interference (RNAi) plants were analysed. Xylem exudates were subjected to speciation analysis and metabolite profiling. Suppression of root nicotianamine synthesis had no effect on Zn and cadmium (Cd) tolerance but rendered plants nickel (Ni)-hypersensitive. It also led to a reduction of Zn root-to-shoot translocation, yet had the opposite effect on Ni mobility, even though both metals form coordination complexes of similar stability with nicotianamine. Xylem Zn concentrations were positively, yet nonstoichiometrically, correlated with nicotianamine concentrations. Two fractions containing Zn coordination complexes were detected in WT xylem. One of them was strongly reduced in AhNAS2-suppressed plants and coeluted with (67) Zn-labelled organic acid complexes. Organic acid concentrations were not responsive to nicotianamine concentrations and sufficiently high to account for complexing the coordinated Zn. We propose a key role for nicotianamine in controlling the efficiency of Zn xylem loading and thereby the formation of Zn coordination complexes with organic acids, which are the main Zn ligands in the xylem but are not rate-limiting for Zn translocation.