Stéphane Mari

Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters

Background Since the identification of the genes controlling the root acquisition of iron (Fe), the control of inter- and intracellular distribution has become an important challenge in understanding metal homeostasis. The identification of the yellow stripe-like (YSL) transporter family has paved the way to decipher the mechanisms of long-distance transport of Fe. Scope Once in the plant, Fe will systematically react with organic ligands whose identity is poorly known so far. Among potential ligands, nicotianamine has been identified as an important molecule for the circulation and delivery of metals since it participates in the loading of copper (Cu) and nickel in xylem and prevents Fe precipitation in leaves. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclusively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe. Maize YS1 is the founding member of a family of membrane transporters called YS1-like (YSL), which functions in root Fe-phytosiderophore uptake from the soil. Next to this well-known Fe acquisition role, most of the other YSL family members are likely to function in plant-wide distribution of metals since (a) they are produced in vascular tissues throughout the plant and (b) they are found in non-Poaceae species that do not synthesize phytosiderophores. The hypothesized activity as Fe-nicotianamine transporters of several YSL members has been demonstrated experimentally by heterologous expression in yeast or by electrophysiology in Xenopus oocytes but, despite numerous attempts, proof of the arabidopsis YSL substrate specificity is still lacking. Reverse genetics, however, has revealed a role for AtYSL members in the remobilization of Cu and zinc from senescing leaves, in the formation of pollen and in the Fe, zinc and Cu loading of seeds. Conclusions Preliminary data on the YSL family of transporters clearly argues in favour of its role in the long-distance transport of metals through and between vascular tissues to eventually support gametogenesis and embryo development.

High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions

This study shows that, in order to acquire manganese when concentrations in the soil are limited, Arabidopsis relies on a root high-affinity manganese uptake system catalyzed by the metal transporter NRAMP1. The finding that overexpression of NRAMP1 produces large plants with increased manganese content paves the way for the biotechnological engineering of plants with improved biomass production. In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis.

The NRAMP6 metal transporter contributes to cadmium toxicity

NRAMP (natural resistance-associated macrophage protein) homologues are evolutionarily conserved bivalent metal transporters. In Arabidopsis, AtNRAMP3 and AtNRAMP4 play a key role in iron nutrition of the germinating plantlet by remobilizing vacuolar iron stores. In the present paper we describe the molecular and physiological characterization of AtNRAMP6. AtNRAMP6 is predominantly expressed in the dry seed embryo and to a lesser extent in aerial parts. Its promoter activity is found diffusely distributed in cotyledons and hypocotyl, as well as in the vascular tissue region of leaf and flower. We show that the AtNRAMP6 transcript coexists with a partially spliced isoform in all shoot cell types tested. When expressed in yeast, AtNRAMP6, but not its misspliced derivative, increased sensitivity to cadmium without affecting cadmium content in the cell. Likewise, Arabidopsis transgenic plants overexpressing AtNRAMP6 were hypersensitive to cadmium, although plant cadmium content remained unchanged. Consistently, a null allele of AtNRAMP6, named nramp6-1, was more tolerant to cadmium toxicity, a phenotype that was reverted by expressing AtNRAMP6 in the mutant background. We used an AtNRAMP6::HA (where HA is haemagglutinin) fusion, shown to be functional in yeast, to demonstrate through immunoblot analysis of membrane fractions and immunofluorescence localization that, in yeast cells, AtNRAMP6 is targeted to a vesicular-shaped endomembrane compartment distinct from the vacuole or mitochondria. We therefore propose that AtNRAMP6 functions as an intracellular metal transporter, whose presence, when modified, is likely to affect distribution/availability of cadmium within the cell.

Identification of the Endodermal Vacuole as the Iron Storage Compartment in the Arabidopsis Embryo

Deciphering how cellular iron (Fe) pools are formed, where they are localized, and which ones are remobilized represents an important challenge to better understand Fe homeostasis. The recent development of imaging techniques, adapted to plants, has helped gain insight into these events. We have analyzed the localization of Fe during embryo development in Arabidopsis (Arabidopsis thaliana) with an improved histochemical staining based on Perls coloration intensified by a second reaction with diaminobenzidine and hydrogen peroxide. The procedure, quick to set up and specific for Fe, was applied directly on histological sections, which dramatically increased its subcellular resolution. We have thus unambiguously shown that in dry seeds Fe is primarily stored in the endodermis cell layer, within the vacuoles, from which it is remobilized during germination. In the vit1-1 mutant, in which the Fe pattern is disturbed, Fe is stored in vacuoles of cortex cells of the hypocotyl/radicle axis and in a single subepidermal cell layer in the cotyledons. During the early stages of embryo development, Fe is evenly distributed in the cells of both wild-type and vit1-1 mutants. Fe eventually accumulates in endodermal cells as the vascular system develops, a process that is impaired in vit1-1. Our results have uncovered a new role for the endodermis in Fe storage in the embryo and have established that the Perls/diaminobenzidine staining is a method of choice to detect Fe in plant tissues and cells.

Nicotianamine over-accumulation confers resistance to nickel in Arabidopsis thaliana.

Nicotianamine is a methionine derivative involved in iron homeostasis, able to bind various other metals in vitro. To investigate its role in vivo, we expressed a nicotianamine synthase cDNA (TcNAS1) isolated from the polymetallic hyperaccumulator Thlaspi caerulescens in Arabidopsis thaliana. Transgenic plants expressing TcNAS1 over-accumulated NA, up to 100-fold more than wild type plants. Furthermore, increased NA levels in different transgenic lines were quantitatively correlated with increased nickel tolerance. The tolerance to nickel is expressed at the cellular level in protoplast experiments and is associated with an increased NA content. We have also shown that the most NA-over accumulating line showed a high tolerance to nickel and a significant Ni accumulation in the leaves when grown on nickel-contaminated soil. Our results highlight a new potential role for nicotianamine in heavy metal tolerance at the cellular but also at the whole plant level, easily transposable to a non-tolerant non-hyperaccumulator species. These results open new perspectives for the modulation of nicotianamine content in plants for phytoremediation.

Speciation of non-covalent nickel species in plant tissue extracts by electrospray Q-TOFMS/MS after their isolation by 2D size exclusion-hydrophilic interaction LC (SEC-HILIC) monitored by ICP-MS

An original approach based on successive size-exclusion and hydrophilic interaction HPLC (HILIC) was developed to purify traces of Ni species from a plant aqueous extract. The degree of purity achieved was for the first time sufficient for the identification, in a natural sample, of a number of non-covalent metal complexes by electrospray Q-TOFMS/MS. Nickel complexes with malate, citrate, histidine, EDTA and nicotianamine (NA) were identified in the roots, xylem, shoots and their protoplasts of a metal hyperaccumulator plant Thlaspi caerulescens. The quantitative recovery of the most stable of these complexes (with EDTA and NA) allowed their quantitative determination by SEC-ICP-MS.

Arabidopsis FERRITIN 1 (AtFer1) gene regulation by the PHOSPHATE STARVATION RESPONSE 1 (AtPHR1) transcription factor reveals a direct molecular link between iron and phosphate homeostasis

Background: Physiological evidences have linked phosphate and iron nutrition in plants. Results: Both PHR1 and PHL1 interact with AtFer1 promoter region and regulate its expression in an iron-independent manner. Conclusion: A molecular link exists between the control of iron and of phosphate homeostasis. Significance: PHR1 and PHL1 play a critical role in the regulation of both phosphate and iron homeostasis. A yeast one-hybrid screening allowed the selection of PHR1 as a factor that interacted with the AtFer1 ferritin gene promoter. In mobility shift assays, PHR1 and its close homologue PHL1 (PHR1-like 1) interact with Element 2 of the AtFer1 promoter, containing a P1BS (PHR1 binding site). In a loss of function mutant for genes encoding PHR1 and PHL1 (phr1 phl1 mutant), the response of AtFer1 to phosphate starvation was completely lost, showing that the two transcription factors regulate AtFer1 expression upon phosphate starvation. This regulation does not involve the IDRS (iron-dependent regulatory sequence) present in the AtFer1 promoter and involved in the iron-dependent regulation. The phosphate starvation response of AtFer1 is not linked to the iron status of plants and is specifically initiated by phosphate deficiency. Histochemical localization of iron, visualized by Perls DAB staining, was strongly altered in a phr1 phl1 mutant, revealing that both PHR1 and PHL1 are major factors involved in the regulation of iron homeostasis.

Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

Background: Iron long distance transport in plants is underdocumented. Results: Iron is delivered to embryos as ferric complexes with citrate/malate. An ascorbate-mediated reduction step is further required to acquire iron. Conclusion: Ascorbate plays a key role for the chemical reduction and transport of Fe2+. Significance: The identification of iron ligands and the transport process is crucial to further understand how iron is distributed within the plant. Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled 55Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.

Plant cell nucleolus as a hot spot for iron.

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

Increased sensitivity to iron deficiency in Arabidopsis thaliana overaccumulating nicotianamine

Nicotianamine (NA) is a non-protein amino acid derivative synthesized from S-adenosyl L-methionine able to bind several metal ions such as iron, copper, manganese, zinc, or nickel. In plants, NA appears to be involved in iron availability and is essential for the plant to complete its biological cycle. In graminaceous plants, NA is also the precursor in the biosynthesis of phytosiderophores. Arabidopsis lines accumulating 4- and 100-fold more NA than wild-type plants were used in order to evaluate the impact of such an NA overaccumulation on iron homeostasis. The expression of iron-regulated genes including the IRT1/FRO2 iron uptake system is highly induced at the transcript level under both iron-sufficient and iron-deficient conditions. Nevertheless, NA overaccumulation does not interfere with the iron uptake mechanisms since the iron levels are similar in the NA-overaccumulating line and wild-type plants in both roots and leaves under both sufficient and deficient conditions. This observation also suggests that the translocation of iron from the root to the shoot is not affected in the NA-overaccumulating line. However, NA overaccumulation triggers an enhanced sensitivity to iron starvation, associated with a decrease in iron availability. This study draws attention to a particular phenotype where NA in excess paradoxically leads to iron deficiency, probably because of an increase of the NA apoplastic pool sequestering iron. This finding strengthens the notion that extracellular NA in the apoplast could be a major checkpoint to control plant iron homeostasis.