Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. Beta-glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues.     

Successful Reproduction Requires the Function of Arabidopsis YELLOW STRIPE-LIKE1 and YELLOW STRIPE-LIKE3 Metal-Nicotianamine Transporters in Both Vegetative and Reproductive Structures

Several members of the Yellow Stripe-Like (YSL) family of proteins are transporters of metals that are bound to the metal chelator nicotianamine or the related set of mugineic acid family chelators known as phytosiderophores. Here, we examine the physiological functions of three closely related Arabidopsis (Arabidopsis thaliana) YSL family members, AtYSL1, AtYSL2, and AtYSL3, to elucidate their role(s) in the allocation of metals into various organs of Arabidopsis. We show that AtYSL3 and AtYSL1 are localized to the plasma membrane and function as iron transporters in yeast functional complementation assays. By using inflorescence grafting, we show that AtYSL1 and AtYSL3 have dual roles in reproduction: their activity in the leaves is required for normal fertility and normal seed development, while activity in the inflorescences themselves is required for proper loading of metals into the seeds. We further demonstrate that the AtYSL1 and AtYSL2 proteins, when expressed from the AtYSL3 promoter, can only partially rescue the phenotypes of a ysl1ysl3 double mutant, suggesting that although these three YSL transporters are closely related and have similar patterns of expression, they have distinct activities in planta. In particular, neither AtYSL1 nor AtYSL2 is able to functionally complement the reproductive defects exhibited by ysl1ysl3 double mutant plants.The transition metals iron (Fe), copper (Cu), and zinc (Zn) are among the most important and most problematic of all the micronutrients used by plants. The importance of these metals stems from their roles as essential cofactors for cellular redox reactions involved in photosynthesis, respiration, and many other reactions. The problematic nature of these metals stems from the same distinct chemical properties that make them so valuable to living systems. These metals, particularly Cu and Fe, are highly reactive and, if overaccumulated, can cause cellular redox damage. Fe presents an additional problem for plants, because it is also only sparingly soluble in aqueous solution and thus is typically not “bioavailable” in soil (Guerinot and Yi, 1994). As a response to these key properties, plants have evolved multifaceted systems to control metal uptake by the root, translocation through the plant body, storage within tissues, and remobilization during reproduction and times of nutrient stress.The nonproteinogenic amino acid nicotianamine (NA) is a strong complexor of various transition metals, particularly Fe(II) (Anderegg and Ripperger, 1989) and Fe(III) (von Wiren et al., 1999), as well as Cu(II), Ni(II), Co(II), Mn(II), and Zn(II) (Anderegg and Ripperger, 1989). NA is present in shoots and roots at concentrations ranging between 20 and 500 nm g⁻¹ fresh weight (Stephan et al., 1990) and is present in both xylem (approximately 20 μm [Pich and Scholz, 1996]) and phloem (approximately 130 μm [Schmidke and Stephan, 1995]), suggesting that it is a major complexor of metals throughout the plant. Much of what we know about NA function in plants comes from studies of a mutant of tomato (Solanum lycopersicum) called chloronerva, in which the single gene encoding NA synthase is disrupted (Herbik et al., 1999; Higuchi et al., 1999; Ling et al., 1999). The chloronerva phenotype is complex. Plants exhibit interveinal chlorosis in young leaves and constitutively activate their root Fe uptake systems, indicating that they have inadequate Fe. However, mature leaves of chloronerva mutants contain excess Fe, implying that the Fe that is present is not being properly localized in the absence of NA. These chloronerva plants also have severe defects in translocation of Cu in the xylem, indicating a clear role for NA in Cu transport. The plants are sterile, indicating that NA is important during plant reproduction. Complementing these classical studies on chloronerva, Takahashi et al. (2003) have developed tobacco (Nicotiana tabacum) plants that heterologously express a barley (Hordeum vulgare) gene encoding the enzyme NA aminotransferase, which converts NA into a nonfunctional intermediate. Recently, the phenotype of quadruple NA synthase mutants was described in Arabidopsis (Arabidopsis thaliana; Klatte et al., 2009). In both studies, the plants exhibited many of the defects caused by the chloronerva mutation, including chlorosis and an array of reproductive abnormalities.Several members of the well-conserved Yellow Stripe-Like (YSL) family of proteins function as metal-NA transporters (DiDonato et al., 2004; Koike et al., 2004; Roberts et al., 2004; Schaaf et al., 2004; Murata et al., 2006; Gendre et al., 2007). The founding member of the YSL family, maize (Zea mays) Yellow Stripe1 (ZmYS1), is the primary means by which roots of grasses take up Fe from the soil. The grasses, a group that includes most of the world’s staple grains (e.g. rice [Oryza sativa], wheat [Triticum aestivum], and maize), use a chelation strategy for primary Fe uptake. In response to Fe starvation, grasses secrete phytosiderophores (PS): derivatives of the mugineic acid family that are structurally similar to NA and that form stable Fe(III) chelates in soil (Tagaki et al., 1984). This accomplishes solubilization of the otherwise nearly insoluble soil Fe. The YS1 protein, located at the root surface, then moves the Fe(III)-PS complexes from the rhizosphere into root cells (Romheld and Marchner, 1986; Curie et al., 2001; Roberts et al., 2004)Arabidopsis has eight YSL genes. Three of these (AtYSL1, At4g24120; AtYSL2, At5g24380; and AtYSL3, At5g53550) are expressed strongly in the xylem parenchyma of leaves and are down-regulated during Fe deficiency (DiDonato et al., 2004; Waters et al., 2006). We have previously shown that double mutant plants with lesions in both AtYSL1 and AtYSL3 display strong interveinal chlorosis. We have hypothesized that the function of these YSL transporters in vegetative tissues is to take up Fe that arrives in leaves via the xylem (Waters et al., 2006). All of the defects displayed by ysl1ysl3 double mutants can be alleviated if excess Fe is applied to the soil, demonstrating that these growth defects are caused primarily by a lack of Fe. Intriguingly, although Fe deficiency appears to be the basis of the double mutant phenotype, the concentrations of several metals are specifically altered in the double mutants (Waters et al., 2006). AtYSL1 single mutant plants have subtle phenotypes, the most striking of which is a decrease in both NA and Fe in seeds (Le Jean et al., 2005). Interestingly, leaves of these mutants contain excess NA, while Fe levels are normal. These observations are consistent with the more obvious and extensive phenotypes exhibited by the ysl1ysl3 double mutant and highlight the idea that AtYSL proteins affect the homeostasis of both Fe and NA.In addition to the vegetative defects mentioned above, the ysl1ysl3 double mutant has multiple defects in reproduction. Double mutant flowers produce few functional pollen grains and thus exhibit greatly reduced fertility. Many of the seeds that these plants do manage to produce are small and contain embryos arrested at various immature stages, which often fail to germinate. These fertility defects can be reversed by application of Fe-ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) solution to the soil, again demonstrating that these growth defects are caused by a lack of Fe (Waters et al., 2006). Expression of YSL1 and YSL3 is very limited in flowers and developing siliques; furthermore, the patterns of expression of YSL1 and YSL3 are distinct and largely nonoverlapping in these structures. However, expression of AtYSL1 and AtYSL3 increases markedly during leaf senescence, a period in which many minerals are remobilized from leaves, presumably for delivery into developing seeds (Himelblau and Amasino, 2001). This model is in good agreement with the accepted model for nutrient loading into seeds proposed originally by Hocking and Pate (1977, 1978), which suggests that metals mobilized from vegetative structures account for 20% to 30% of the content in seeds. Direct measurements of metals in senescing and younger leaves demonstrated that double mutants failed to mobilize Zn and Cu from leaves. Seeds produced by the double mutant plants contained reduced levels of Zn and Cu, the same metals that failed to be mobilized out of the leaves (Waters et al., 2006). This led us to propose a model in which the activity of AtYSL1 and AtYSL3 in leaves was required for correct localization of metals into the seeds. However, seeds also had low Fe levels, even though Fe appeared to be mobilized normally from leaves of the double mutants.Here, we further investigate the role(s) of AtYSL1 and AtYSL3 in the allocation of metals into various organs of Arabidopsis. AtYSL1 and AtYSL3 are localized to the plasma membrane, and each is capable of suppressing the growth defect of yeast lacking normal Fe uptake, indicating that the most likely biochemical function for these proteins is in uptake of Fe(II)-NA complexes. We have used inflorescence grafting to determine the relative roles of AtYSL1 and AtYSL3 in leaves and inflorescences during seed development. These proteins are found to have dual roles: activity in the leaves is required for normal inflorescence development, while activity in the inflorescences themselves is required for proper loading of metals into the seeds. We have further examined the effect of overexpressing AtYSL3, which resulted in a small increase in Cu in shoots, and have demonstrated that the AtYSL1 protein, when expressed from the AtYSL3 promoter, can only partially rescue the phenotypes of the ysl1ysl3 double mutant, indicating that these proteins have distinct biochemical activities. A third AtYSL from the same subgroup of the YSL family, AtYSL2, also only partially complements the phenotypes of ysl1ysl3 double mutants, suggesting that although these three YSL transporters are closely related, they have distinct activities in planta.     

Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3

Minimal information exists on whole-plant dynamics of mineral flow through Arabidopsis thaliana or on the source tissues responsible for mineral export to developing seeds. Understanding these phenomena in a model plant could help in the development of nutritionally enhanced crop cultivars. A whole-plant partitioning study, using sequential harvests, was conducted to characterize growth and mineral concentrations and contents of rosettes, cauline leaves, stems, immature fruit, mature fruit hulls, and seeds of three WT lines (Col-0, Ler, and Cvi) and one mutant line (Col-0::ysl1ysl3). Shoot mineral content increased throughout the life cycle for all minerals, although tissue-specific mineral partitioning differed between genotypes. In particular, iron (Fe), zinc (Zn), and copper (Cu) were aberrantly distributed in ysl1ysl3. Remobilization was observed for several minerals from various tissues, including cauline leaves and silique hulls, but the amounts were generally far below the total mineral accretion observed in seeds. When YSL1 and YSL3 are nonfunctional, Cu, Fe, and Zn are not effectively remobilized from, or do not effectively pass through, leaf and maternal fruit tissues. With respect to seed mineral accretion in Arabidopsis, continued uptake and translocation of minerals to source tissues during seed fill are as important, if not more important, than remobilization of previously stored minerals.     

Arabidopsis SUMO E3 ligase SIZ1 is involved in excess copper tolerance

The reversible conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates occurs as a posttranslational regulatory process in eukaryotic organisms. In Arabidopsis (Arabidopsis thaliana), several stress-responsive SUMO conjugations are mediated mainly by the SUMO E3 ligase SIZ1. In this study, we observed a phenotype of hypersensitivity to excess copper in the siz1-2 and siz1-3 mutants. Excess copper can stimulate the accumulation of SUMO1 conjugates in wild-type plants but not in the siz1 mutant. Copper accumulated to a higher level in the aerial parts of soil-grown plants in the siz1 mutant than in the wild type. A dramatic difference in copper distribution was also observed between siz1 and wild-type Arabidopsis treated with excess copper. As a result, the shoot-to-root ratio of copper concentration in siz1 is nearly twice as high as that in the wild type. We have found that copper-induced Sumoylation is involved in the gene regulation of metal transporters YELLOW STRIPE-LIKE 1 (YSL1) and YSL3, as the siz1 mutant is unable to down-regulate the expression of YSL1 and YSL3 under excess copper stress. The hypersensitivity to excess copper and anomalous distribution of copper observed in the siz1 mutant are greatly diminished in the siz1ysl3 double mutant and slightly in the siz1ysl1 double mutant. These data suggest that SIZ1-mediated sumoylation is involved specifically in copper homeostasis and tolerance in planta.     

Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis

Ferritin protein nanocages are the main iron store in mammals. They have been predicted to fulfil the same function in plants but direct evidence was lacking. To address this, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. Loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron, as shown by reduced growth and strong defects in flower development. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporters genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, we show that, in the absence of ferritin, plants have higher levels of reactive oxygen species, and increased activity of enzymes involved in their detoxification. Seed germination also showed higher sensitivity to pro-oxidant treatments. Arabidopsis ferritins are therefore essential to protect cells against oxidative damage.     

The Analysis of Arabidopsis Nicotianamine Synthase Mutants Reveals Functions for Nicotianamine in Seed Iron Loading and Iron Deficiency Responses

Nicotianamine chelates and transports micronutrient metal ions in plants. It has been speculated that nicotianamine is involved in seed loading with micronutrients. A tomato (Solanum lycopersicum) mutant (chloronerva) and a tobacco (Nicotiana tabacum) transgenic line have been utilized to analyze the effects of nicotianamine loss. These mutants showed early leaf chlorosis and had sterile flowers. Arabidopsis (Arabidopsis thaliana) has four NICOTIANAMINE SYNTHASE (NAS) genes. We constructed two quadruple nas mutants: one had full loss of NAS function, was sterile, and showed a chloronerva-like phenotype (nas4x-2); another mutant, with intermediate phenotype (nas4x-1), developed chlorotic leaves, which became severe upon transition from the vegetative to the reproductive phase and upon iron (Fe) deficiency. Residual nicotianamine levels were sufficient to sustain the life cycle. Therefore, the nas4x-1 mutant enabled us to study late nicotianamine functions. This mutant had no detectable nicotianamine in rosette leaves of the reproductive stage but low nicotianamine levels in vegetative rosette leaves and seeds. Fe accumulated in the rosette leaves, while less Fe was present in flowers and seeds. Leaves, roots, and flowers showed symptoms of Fe deficiency, whereas leaves also showed signs of sufficient Fe supply, as revealed by molecular-physiological analysis. The mutant was not able to fully mobilize Fe to sustain Fe supply of flowers and seeds in the normal way. Thus, nicotianamine is needed for correct supply of seeds with Fe. These results are fundamental for plant manipulation approaches to modify Fe homeostasis regulation through alterations of NAS genes.     

Increased nicotianamine biosynthesis confers enhanced tolerance of high levels of metals, in particular nickel, to plants

Nicotianamine, a plant-derived chelator of metals, is produced by the trimerization of S-adenosylmethionine catalyzed by nicotianamine synthase. We established transgenic Arabidopsis and tobacco plants that constitutively overexpress the barley nicotianamine synthase gene. Nicotianamine synthase overexpression resulted in increased biosynthesis of nicotianamine in transgenic plants, which conferred enhanced tolerance of high levels of metals, particularly nickel, to plants. Promoter activities of four nicotianamine synthase genes in Arabidopsis were all increased in response to excess nickel, suggesting that nicotianamine plays an important role in the detoxification of nickel in plants. Furthermore, transgenic tobacco plants with a high level of nicotianamine grew well in a nickel-enriched serpentine soil without developing any symptoms of nickel toxicity. Our results indicate that nicotianamine plays a critical role in metal detoxification, and this can be a powerful tool for use in phytoremediation.     

Put the metal to the petal: metal uptake and transport throughout plants

Compared to other organisms, plants have expanded families of transporters that are involved in the uptake and efflux of metals. Fortunately, in many cases, the examination of double mutants has been sufficient to overcome the challenge of studying functionally redundant gene families. Plants that lack two heavy-metal-transporting P-type ATPase family members (HMA2 and HMA4) reveal a function for these transporters in Zn translocation from roots to shoots. Likewise, the phenotype of plants that lack two natural resistance associated macrophage protein (NRAMP) homologs (NRAMP3 and NRAMP4) implicate these metal uptake proteins in the mobilization of vacuolar Fe stores during seed germination. Most families of metal transporters are ubiquitous but the Yellow Stripe1-Like (YSL) family is plant specific and YSL family members have been implicated in the transport of metals that are complexed with a plant specific chelator called nicotianamine (NA).     

Investigation of ascorbate-mediated iron release from ferric phytosiderophores in the presence of nicotianamine

Phytosiderophores (PS) are strong iron chelators, produced by graminaceous plants under iron deficiency. The ability of released PS to chelate iron(III), and subsequent uptake of this chelate into roots by YS1-type transport proteins, are well-known. The mechanism of iron release from the stable chelate inside the plant cell, however, is unclear. One possibility involves the reduction of ferric PS in the presence of an iron(II) chelator via ternary complex formation. Here, the conversion of ferric PS species by ascorbate in the presence of the intracellular ligand nicotianamine (NA) has been investigated at cytosolic pH (pH 7.3), leading to the formation of a ferrous NA chelate. This reaction takes place when supplying Fe(III) as a chelate with 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), with the reaction rate decreasing in this order. The progress of the conversion of ferric DMA to ferrous NA was monitored in real-time by high resolution mass spectrometry (FTICR-MS), and the results are complemented by electrochemical measurements (cyclic voltammetry), which allows detecting reactive intermediates and their change with time at high sensitivity. Hence, the combined results of electrochemistry and mass spectrometry indicate an ascorbate-mediated mechanism for the iron release from ferric PS, which highlights the role of ascorbate as a simple, but effective plant reductant.     

Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis

The essential micronutrients Fe and Zn often limit plant growth but are toxic in excess. Arabidopsis thaliana ZINC-INDUCED FACILITATOR1 (ZIF1) is a vacuolar membrane major facilitator superfamily protein required for basal Zn tolerance. Here, we show that overexpression of ZIF1 enhances the partitioning into vacuoles of the low molecular mass metal chelator nicotianamine and leads to pronounced nicotianamine accumulation in roots, accompanied by vacuolar buildup of Zn. Heterologous ZIF1 protein localizes to vacuolar membranes and enhances nicotianamine contents of yeast cells engineered to synthesize nicotianamine, without complementing a Zn-hypersensitive mutant that additionally lacks vacuolar membrane Zn(2+)/H(+) antiport activity. Retention in roots of Zn, but not of Fe, is enhanced in ZIF1 overexpressors at the expense of the shoots. Furthermore, these lines exhibit impaired intercellular Fe movement in leaves and constitutive Fe deficiency symptoms, thus phenocopying nicotianamine biosynthesis mutants. Hence, perturbing the subcellular distribution of the chelator nicotianamine has profound, yet distinct, effects on Zn and Fe with respect to their subcellular and interorgan partitioning. The zif1 mutant is also hypersensitive to Fe deficiency, even in media lacking added Zn. Therefore, accurate levels of ZIF1 expression are critical for both Zn and Fe homeostasis. This will help to advance the biofortification of crops.     

Bio-available zinc in rice seeds is increased by activation tagging of nicotianamine synthase

We generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2'-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2.     

Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis

The metal chelator nicotianamine promotes the bioavailability of Fe and reduces cellular Fe toxicity. For breeding Fe-efficient crops, we need to explore the fundamental impact of nicotianamine on plant development and physiology. The quadruple nas4x-2 mutant of Arabidopsis thaliana cannot synthesize any nicotianamine, shows strong leaf chlorosis, and is sterile. To date, these phenotypes have not been fully explained. Here, we show that sink organs of this mutant were Fe deficient, while aged leaves were Fe sufficient. Upper organs were also Zn deficient. We demonstrate that transport of Fe to aged leaves relied on citrate, which partially complemented the loss of nicotianamine. In the absence of nicotianamine, Fe accumulated in the phloem. Our results show that rather than enabling the long-distance movement of Fe in the phloem (as is the case for Zn), nicotianamine facilitates the transport of Fe from the phloem to sink organs. We delimit nicotianamine function in plant reproductive biology and demonstrate that nicotianamine acts in pollen development in anthers and pollen tube passage in the carpels. Since Fe and Zn both enhance pollen germination, a lack of either metal may contribute to the reproductive defect. Our study sheds light on the physiological functions of nicotianamine.     

Yellow stripe1. Expanded roles for the maize iron-phytosiderophore transporter

Graminaceous monocots, including most of the world's staple grains (i.e. rice, corn, and wheat) use a chelation strategy (Strategy II) for primary acquisition of iron from the soil. Strategy II plants secrete phytosiderophores (PS), compounds of the mugineic acid family that form stable Fe(III) chelates in soil. Uptake of iron-PS chelates, which occurs through specific transporters at the root surface, thus represents the primary route of iron entry into Strategy II plants. The gene Yellow stripe1 (Ys1) encodes the Fe(III)-PS transporter of maize (Zea mays). Here the physiological functions performed by maize YS1 were further defined by examining the pattern of Ys1 mRNA and protein accumulation and by defining YS1 transport specificity in detail. YS1 is able to translocate iron that is bound either by PS or by the related compound, nicotianamine; thus, the role of YS1 may be to transport either of these complexes. Ys1 expression at both the mRNA and protein levels responds rapidly to changes in iron availability but is not strongly affected by limitation of copper or zinc. Our data provide no support for the idea that YS1 is a transporter of zinc-PS, based on YS1 biochemical activity and Ys1 mRNA expression patterns in response to zinc deficiency. YS1 is capable of transporting copper-PS, but expression data suggest that the copper-PS uptake has limited significance in primary uptake of copper.     

Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters

Background and Aims

Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency.

Methods

The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography.

Key Results

Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated.

Conclusions

PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis.     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains

Key message

Rice OsYSL9 is a novel transporter for Fe(II)-nicotianamine and Fe(III)-deoxymugineic acid that is responsible for internal iron transport, especially from endosperm to embryo in developing seeds.

Abstract

Metal chelators are essential for safe and efficient metal translocation in plants. Graminaceous plants utilize specific ferric iron chelators, mugineic acid family phytosiderophores, to take up sparingly soluble iron from the soil. Yellow Stripe 1-Like (YSL) family transporters are responsible for transport of metal-phytosiderophores and structurally similar metal-nicotianamine complexes. Among the rice YSL family members (OsYSL) whose functions have not yet been clarified, OsYSL9 belongs to an uncharacterized subgroup containing highly conserved homologs in graminaceous species. In the present report, we showed that OsYSL9 localizes mainly to the plasma membrane and transports both iron(II)-nicotianamine and iron(III)-deoxymugineic acid into the cell. Expression of OsYSL9 was induced in the roots but repressed in the nonjuvenile leaves in response to iron deficiency. In iron-deficient roots, OsYSL9 was induced in the vascular cylinder but not in epidermal cells. Although OsYSL9-knockdown plants did not show a growth defect under iron-sufficient conditions, these plants were more sensitive to iron deficiency in the nonjuvenile stage compared with non-transgenic plants. At the grain-filling stage, OsYSL9 expression was strongly and transiently induced in the scutellum of the embryo and in endosperm cells surrounding the embryo. The iron concentration was decreased in embryos of OsYSL9-knockdown plants but was increased in residual parts of brown seeds. These results suggested that OsYSL9 is involved in iron translocation within plant parts and particularly iron translocation from endosperm to embryo in developing seeds.

     

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

Background: Since the identification of the genes controlling the root acquisitionof iron (Fe), the control of inter- and intracellular distributionhas become an important challenge in understanding metal homeostasis.The identification of the yellow stripe-like (YSL) transporterfamily has paved the way to decipher the mechanisms of long-distancetransport of Fe. Scope: Once in the plant, Fe will systematically react with organicligands whose identity is poorly known so far. Among potentialligands, nicotianamine has been identified as an important moleculefor the circulation and delivery of metals since it participatesin the loading of copper (Cu) and nickel in xylem and preventsFe precipitation in leaves. Nicotianamine is a precursor ofphytosiderophores, which are high-affinity Fe ligands exclusivelysynthesized by Poaceae species and excreted by roots for thechelation and acquisition of Fe. Maize YS1 is the founding memberof a family of membrane transporters called YS1-like (YSL),which functions in root Fe–phytosiderophore uptake fromthe soil. Next to this well-known Fe acquisition role, mostof the other YSL family members are likely to function in plant-widedistribution of metals since (a) they are produced in vasculartissues throughout the plant and (b) they are found in non-Poaceaespecies that do not synthesize phytosiderophores. The hypothesizedactivity as Fe–nicotianamine transporters of several YSLmembers has been demonstrated experimentally by heterologousexpression in yeast or by electrophysiology in Xenopus oocytesbut, despite numerous attempts, proof of the arabidopsis YSLsubstrate specificity is still lacking. Reverse genetics, however,has revealed a role for AtYSL members in the remobilizationof Cu and zinc from senescing leaves, in the formation of pollenand in the Fe, zinc and Cu loading of seeds. Conclusions: Preliminary data on the YSL family of transporters clearly arguesin favour of its role in the long-distance transport of metalsthrough and between vascular tissues to eventually support gametogenesisand embryo development.     

Nicotianamine Over-accumulation Confers Resistance to Nickel in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

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.