Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in zinc hyperaccumulation

Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels >100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.     

Complexation and detoxification of Zn and Cd in metal accumulating plants

Metal accumulating plants exposed to toxic levels of zinc (Zn) and cadmium (Cd) uptake metals through extracellular and intracellular complexation with inorganic and organic ligand formation. However, little is known about the nature and formation mechanism of these metal–ligand complexes. Though, Zn and Cd have many similar chemical properties, yet their complexation and compartmentalization in plants vary with plant species. In principal, the question arises what factors govern Zn and Cd partitioning in plants? What form of the metal is taken up by the root, and is further distributed and accumulated in both vegetative and reproductive tissues? Therefore, the aim of present study is to address several questions concerning the mechanisms of Zn and Cd coordination and compartmentalization in plants using X-ray absorption spectroscopy (XAS) technique. XAS allows direct determination of elemental oxidation states and coordination environments in different plant tissues. This review article briefly explains some other important techniques of XAS; EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near edge structure), which are employed for determining Zn and Cd complexation within the plant. Therefore, In present review, the predominant as well as the minor chemical forms of Zn and Cd present in particular plant tissue have been discussed which could give better insight towards metal accumulation and detoxification mechanisms operated in plants. This information could assist in employing suitable hyperaccumulator plants for metal phytoextraction and reclamation of metal contaminated sites.     

Identification and molecular characterization of the nicotianamine synthase gene family in bread wheat

Nicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.     

Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions

Nicotianamine (NA) is an intermediate in the biosynthetic pathway of the mugineic acid family phytosiderophores (MAs), which are crucial components of the iron acquisition apparatus of graminaceous plants. In non-graminaceous plants, NA is thought to be an essential chelator for metal cation homeostasis. Thus NA plays a key role in Fe metabolism and homeostasis in all higher plants. Nicotianamine synthase (NAS, EC 2.5.1.43) catalyzes the trimerization of S-adenosylmethionine to form one molecule of NA. Barley, a plant that is resistant to Fe deficiency, secretes large amounts of MAs, whereas rice, a plant that is susceptible to Fe deficiency, secretes only small amounts. In this study we isolated a genomic fragment containing HvNAS1 from barley and three rice cDNA clones, osnas1, osnas2 and osnas3, from Fe-deficient rice roots. We also isolated a genomic fragment containing both OsNAS1 and OsNAS2. In contrast to barley, in which Fe deficiency induces the expression of NAS genes only in roots, Fe deficiency in rice induced NAS gene expression in both roots and chlorotic leaves. The amounts of endogenous NA in both the roots and leaves were higher than in barley. We introduced barley genomic DNA fragments containing HvNAS1 with either 9 or 2 kb of the 5'-flanking region into rice, using Agrobacterium-mediated transformation. Fe deficiency induced HvNAS1 expression in both roots and leaves of the transgenic rice, as occurs with rice NAS genes. Barley and rice NAS genes are compared in a discussion of alteration of the NAS genes during adaptation to Fe deficiency.     

Nicotianamine is a major player in plant Zn homeostasis

Nicotianamine (NA) is among the most studied plant metal chelators. A large body of evidence supports its crucial role for Fe distribution in plants and as a precursor of phytosiderophore synthesis in grasses. NA forms stable complexes in vitro not only with Fe(II) and Fe(III) but also with various other divalent metal cations including Zn(II). Early observations indicated a possible contribution of NA to Zn trafficking in plants. Numerous studies on transgenic monocot and dicot plants with modulated NA levels have since then reported Zn accumulation phenotypes. NAS genes were shown to represent promising targets for biofortification efforts. For instance, NA was found to bind Zn in rice grains in a form bioavailable for humans. Recently, additional strong support for the existence of Zn–NA complexes in planta has been obtained in rice, Arabidopsis thaliana and the Zn hyperaccumulating plant A. halleri. We review the evidence for a role of NA in the intercellular and long-distance transport of Zn in plants and discuss open questions.     

Overexpression of a <Emphasis Type="Italic">Miscanthus sacchariflorus</Emphasis> yellow stripe-like transporter <Emphasis Type="Italic">MsYSL1</Emphasis> enhances resistance of <Emphasis Type="Italic">Arabidopsis</Emphasis> to cadmium by mediating metal ion reallocation

The yellow stripe-like (YSL) family of transporters mediates the uptake, translocation, and distribution of various mineral elements in vivo by transferring metal ions chelated with phytosiderophore or nicotianamine (NA). However, little is known about the roles of the YSL genes against cadmium in planta. In this study, we first cloned and characterized a vital member of the YSL gene family, MsYSL1, from the bioenergy plant Miscanthus sacchariflorus. MsYSL1 localized in the plasma membrane and was widely expressed throughout the whole seedling with the highest expression level in the stem. In addition, its expression in the root was stimulated by excess manganese (Mn), cadmium (Cd), and lead, and a shortage of iron (Fe), zinc (Zn), and copper. Functional complementation in yeast indicated that MsYSL1 showed transport activity for Fe(II)–NA and Zn–NA, but not for Cd–NA. Although they exhibited no significant differences versus the wild type under normal cultivation conditions, MsYSL1-overexpressing Arabidopsis lines displayed a higher resistance to Cd accompanied by longer root lengths, lower Cd, Zn, and Mn levels in roots, and higher Cd, Fe, and Mn translocation ratios under Cd stress. Moreover, genes related to NA synthesis, metal translocation, long-distance transport, and Cd exclusion were highly induced in transgenic lines under Cd stress. Thus, MsYSL1 may be an essential transporter for diverse metal–NAs to participate in the Cd detoxification by mediating the reallocation of other metal ions.     

Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens

Plant metal hyperaccumulator species are widely used as models to unravel the heavy metal tolerance and hyperaccumulation mechanisms. Thlaspi caerulescens is capable of tolerating and hyperaccumulating Zn, Cd, and Ni. A search for factors involved in the cellular tolerance to Ni, based on yeast screens, led to isolation of a cDNA encoding a functional nicotianamine (NA) synthase (NAS). The T. caerulescens genome appears to contain a single copy of the NAS gene named TcNAS whose expression is restricted to the leaves. The analysis of dose-response and time-course Ni treatments have revealed that the exposure to Ni triggers the accumulation of NA in the roots. Because neither TcNAS expression nor NAS activity were detected in the roots, the NA accumulation in roots is most probably the result of its translocation from the leaves. Once in the roots, NA, together with Ni, is subsequently found in the xylem, for redirection to the aerial parts. Using liquid chromatography coupled to inductively coupled plasma or electrospray ionization mass spectrometry, it has been shown that part of the Ni is translocated as a stable Ni-NA complex in the xylem sap. This circulation of NA, Ni, and NA-Ni chelates is absent in the non-tolerant non-hyperaccumulator related species T. arvense. Taken together, the results provide direct physiological and chemical evidence for NA and NA-heavy metal complex translocation in a hyperaccumulator species.     

TcYSL3, a member of the YSL gene family from the hyper-accumulator Thlaspi caerulescens, encodes a nicotianamine-Ni/Fe transporter

The two main features of plant hyper-accumulator species are the massive translocation of heavy metal ions to the aerial parts and their tolerance to such high metal concentrations. Recently, several lines of evidence have indicated a role for nicotianamine (NA) in metal homeostasis, through the chelation and transport of NA-metal complexes. The function of transport of NA-metal chelates, required for the loading and unloading of vessels, has been assigned to the Yellow Stripe 1 (YSL)-Like family of proteins. We have characterized three YSL genes in Thlaspi caerulescens in the context of hyper-accumulation. The three YSL genes are expressed at high rates compared with their Arabidopsis thaliana homologs but with distinct patterns. While TcYSL7 was highly expressed in the flowers, TcYSL5 was more highly expressed in the shoots, and the expression of TcYSL3 was equivalent in all the organs tested. In situ hybridizations have shown that TcYSL7 and TcYSL5 are expressed around the vasculature of the shoots and in the central cylinder in the roots. The exposure to heavy metals (Zn, Cd, Ni) does not affect the high and constitutive expression of the TcYSL genes. Finally, we have demonstrated by mutant yeast complementation and uptake measurements that TcYSL3 is an Fe/Ni-NA influx transporter. This work provides therefore molecular, histological and biochemical evidence supporting a role for YSL transporters in the overall scheme of NA and NA-metal, particularly NA-Ni, circulation in a metal hyper-accumulator plant.     

Zinc modulates copper coordination mode in prion protein octa-repeat subdomains

In this work we present and analyse XAS measurements carried out on various portions of Prion-protein tetra-octa-repeat peptides in complexes with Cu(II) ions, both in the presence and in the absence of Zn(II). Because of the ability of the XAS technique to provide detailed local structural information, we are able to demonstrate that Zn acts by directly interacting with the peptide, in this way competing with Cu for binding with histidine. This finding suggests that metal binding competition can be important in the more general context of metal homeostasis.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn(2+)-ATPases

Zn(2+) is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn(2+) homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn(2+) at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn(2+)-ATPase responsible for intracellular Zn(2+) efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal-ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)(2)S(2) that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn(2+) bound to the TM-MBS of P-type ATPase transporters.     

No metal cofactor in orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) is an important enzyme that catalyzes the final step of de novo pyrimidine nucleotide biosynthesis. The mechanism of this unique enzyme and whether metal ions play any role in catalysis have been topics of intense research interest. In this report, the role of Zn in ODCase was reexamined. Atomic absorption (AA) and X-ray absorption (XAS) spectroscopic studies did not detect zinc in active enzyme samples at high concentration. The XAS results also indicated the absence of other transition metal ions in ODCase.     

Iron-dependent changes of heavy metals,nicotianamine, and citrate in different plant organs and in the xylem exudate of two tomato genotypes. Nicotianamine as possible copper translocator

The influence of Fe nutrition on the distribution of the heavy metals Fe, Mn, Zn, and Cu and of the heavy metal chelators nicotianamine (NA) and citrate in 6 different shoot and 3 different root parts and in xylem exudate of a NA-containing tomato wild type and its NA-less mutant was investigated. Under the same Fe supply the mutant showed higher Fe, Mn, and Zn concentrations in all organs investigated, with exception of the shoot apex. The Cu concentration in the mutant was only in root parts higher than in the wild type but much lower in leaves. Analyses of xylem exudate showed that Fe, Mn, and Zn were readily translocated by both genotypes from the roots to the shoot at all levels of Fe supply, whereas in the absence of NA, Cu was only poorly transported. Citrate as main Fe chelator in the xylem was present in high concentrations in xylem exudate of the wild type under low Fe supply but in the mutant also at 10 M FeEDTA. NA occurred in xylem exudate of the wild type in concentrations high enough to chelate heavy metal ions.Generally, high Fe supply induced a decrease of Mn, Cu, and Zn concentrations in all organs of the wild type whereas high concentrations were observed in most cases under Fe deficiency. A positive correlation between Fe supply and NA concentration existed only in the shoot apex and in the xylem exudate of wild type plants. From the correlation between Cu and NA translocation and from the high stability constant of the NA-Cu-complex (log K=18.6) it is concluded that NA is a chelator for Cu in the xylem, whereas the translocation of Fe, Mn, and Zn is independent of NA.     

Transporters of ligands for essential metal ions in plants

Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.     

Relationships of nicotianamine and other amino acids with nickel, zinc and iron in Thlaspi hyperaccumulators

Experimental evidence suggests that nicotianamine (NA) is involved in the complexation of metal ions in some metal-hyperaccumulating plants. Closely-related nickel (Ni)- and zinc (Zn)-hyperaccumulating species were studied to determine whether a correlation exists between the Ni and Zn concentrations and NA in foliar tissues. A liquid chromatography-mass spectrometry (LC-MS) procedure was developed to quantify the NA and amino acid contents using the derivatizing agent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. A strong correlation emerged between Ni and NA, but not between Zn and NA. Concentrations of NA and L-histidine (His) also increased in response to higher Ni concentrations in the hydroponic solution supplied to a serpentine population of Thlaspi caerulescens. An inversely proportional correlation was found between the iron (Fe) and Ni concentrations in the leaves. Correlations were also found between Zn and asparagine. The results obtained in this study suggest that NA is involved in hyperaccumulation of Ni but not Zn. The inverse proportionality between the Ni and Fe concentrations in the leaf may suggest that Ni and Fe compete for complexation to NA.     

Synthesis and proof-of-function of a [14C]-labelled form of the plant iron chelator nicotianamine using recombinant nicotianamine synthase from barley

The amino acid nicotianamine (NA) is essential for micronutrient metabolism in plants. Lack of NA results in a chlorotic phenotype and oxidative stress, since NA is a chelator of iron and other metal nutrients. To investigate the precise cellular function of NA in micronutrient transport and homeostasis, a protocol for the production of [¹⁴C]-labelled NA was developed. Recombinant NA synthase was used to generate [¹⁴C]-NA from [¹⁴C]- S -adenosylmethionine. After purification by solid-phase ion exchange about 66% yield was achieved. The identity of the [¹⁴C]-NA with chemically synthesized NA was demonstrated by several independent methods, including two TLC systems, two HPLC systems and immuno-detection. Moreover, biological function was shown by complementation of the Lycopersicon esculentum mutant chloronerva that is free of NA due to a defect in NA synthase. Proof-of-function for the produced [¹⁴C]-NA as a suitable tool for transport studies was provided monitoring the distribution of [¹⁴C]-NA after feeding to tomato and Ricinus communis seedlings.     

Using synchrotron X-ray fluorescence microprobes in the study of metal homeostasis in plants

Background and Aims

This Botanical Briefing reviews the application of synchrotron X-ray fluorescence (SXRF) microprobes to the plant sciences; how the technique has expanded our knowledge of metal(loid) homeostasis, and how it can be used in the future.

Scope

The use of SXRF microspectroscopy and microtomography in research on metal homeostasis in plants is reviewed. The potential use of SXRF as part of the ionomics toolbox, where it is able to provide fundamental information on the way that plants control metal homeostasis, is recommended.

Conclusions

SXRF is one of the few techniques capable of providing spatially resolved in-vivo metal abundance data on a sub-micrometre scale, without the need for chemical fixation, coating, drying or even sectioning of samples. This gives researchers the ability to uncover mechanisms of plant metal homeostasis that can potentially be obscured by the artefacts of sample preparation. Further, new generation synchrotrons with smaller beam sizes and more sensitive detection systems will allow for the imaging of metal distribution within single living plant cells. Even greater advances in our understanding of metal homeostasis in plants can be gained by overcoming some of the practical boundaries that exist in the use of SXRF analysis.Key words: Metal homeostasis, synchrotron X-ray fluorescence, SXRF, microspectroscopy, microtomography, X-ray absorption spectroscopy, XAS, ionomics, Arabidopsis thaliana, hyperaccumulator     

The expression of heterologous Fe (III) phytosiderophore transporter HvYS1 in rice increases Fe uptake,translocation and seed loading and excludes heavy metals by selective Fe transport

Many metal transporters in plants are promiscuous, accommodating multiple divalent cations including some which are toxic to humans. Previous attempts to increase the iron (Fe) and zinc (Zn) content of rice endosperm by overexpressing different metal transporters have therefore led unintentionally to the accumulation of copper (Cu), manganese (Mn) and cadmium (Cd). Unlike other metal transporters, barley Yellow Stripe 1 (HvYS1) is specific for Fe. We investigated the mechanistic basis of this preference by constitutively expressing HvYS1 in rice under the control of the maize ubiquitin1 promoter and comparing the mobilization and loading of different metals. Plants expressing HvYS1 showed modest increases in Fe uptake, root‐to‐shoot translocation, seed accumulation and endosperm loading, but without any change in the uptake and root‐to‐shoot translocation of Zn, Mn or Cu, confirming the selective transport of Fe. The concentrations of Zn and Mn in the endosperm did not differ significantly between the wild‐type and HvYS1 lines, but the transgenic endosperm contained significantly lower concentrations of Cu. Furthermore, the transgenic lines showed a significantly reduced Cd uptake, root‐to‐shoot translocation and accumulation in the seeds. The underlying mechanism of metal uptake and translocation reflects the down‐regulation of promiscuous endogenous metal transporters revealing an internal feedback mechanism that limits seed loading with Fe. This promotes the preferential mobilization and loading of Fe, therefore displacing Cu and Cd in the seed.