Suberoylanilide hydroxamic acid, a potent histone deacetylase inhibitor; its X-ray crystal structure and solid state and solution studies of its Zn(II), Ni(II), Cu(II) and Fe(III) complexes

Reaction of the potent hydroxamate-based histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), with hydrated metal salts of Fe(III), Cu(II), Ni(II) and Zn(II) yielded a tris-hydroxamato complex in the case of Fe(III) and bis-hydroxamato complexes in the case of Cu(II), Ni(II) and Zn(II) both in the solid state and in solution. Reaction of the secondary hydroxamic acid, N-Me-SAHA, also yielded a tris-hydroxamato complex in the case of Fe(III) and bis-hydroxamato complexes in the case of Cu(II), Ni(II) and Zn(II) in solution. These metal complexes have the hydroxamato moiety coordinated in an O,O’-bidentate fashion. Stability constants of the metal complexes formed with SAHA and N-Me-SAHA in a DMSO/H₂O 70/30%(v/v) mixture are described. A novel crystal structure of SAHA together with a novel synthesis for N-Me-SAHA are also reported.     

Nicotianamine forms complexes with Zn(II) in vivo

The non-proteinogenic amino acid nicotianamine (NA) is a major player in plant metal homeostasis. It is known to form complexes with different transition metals in vitro. Available evidence associates NA with translocation of Fe, and possibly other micronutrients, to and between different plant cells and tissues. To date, however, it is still extremely challenging to detect metal-ligand complexes in vivo because tissue disruption immediately changes the chemical environment and thereby the availability of binding partners. In order to overcome this limitation we used various Schizosaccharomyces pombe strains expressing a plant NAS gene to study formation of metal-NA complexes in vivo. Tolerance, accumulation and competition data clearly indicated formation of Zn(ii)-NA but not of Cu(ii)-NA complexes. Zn(ii)-NA was then identified by X-ray absorption spectroscopy (XAS). About half of the cellular Zn was found to be bound by NA in NAS-expressing cells while no NA-like ligands were detected by XAS in control cells not expressing NAS. Given the high conservation of eukaryotic metal homeostasis components, these results strongly suggest the possible existence of Zn(ii)-NA complexes also in planta. Reported observations implicating NA in plant Zn homeostasis would then indeed be attributable to direct interaction of Zn(ii) with NA rather than only indirectly to perturbations in Fe metabolism. Re-evaluation of extended X-ray absorption fine structure (EXAFS) spectra for the Zn hyperaccumulator Thlaspi caerulescens showed that NA is as expected not a major storage ligand for Zn. Instead it is hypothesized to be involved in efficient translocation of Zn to above-ground tissues in hyperaccumulators.     

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.     

Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco

Key message

BjYSL7 encodes a plasma-localized metal–NA transporter and has transport Fe(II)–NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots.

Abstract

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO₄, NiCl₂, and CdCl₂ stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)–NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl₂ and NiCl₂. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.     

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.     

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine (NA) and 2′‐deoxymugineic acid (DMA) – that play key roles in metal transport and nutrition. The CE‐OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE‐OsNAS2 grain and positively correlated with NA and DMA concentrations.     

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.     

Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less ⁶²Zn-DMA than ⁶²Zn²⁺. Importantly, supply of ⁶²Zn-DMA rather than ⁶²Zn²⁺ increased the translocation of ⁶²Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Motofumi Suzuki and Takashi Tsukamoto equally contributed to this work.     

Kinetics and thermodynamics of metal‐binding to histone deacetylase 8

Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)-dependent deacetylase on the basis of Zn(II)-dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)-HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein-labeled suberoylanilide hydroxamic acid (fl-SAHA). fl-SAHA binds specifically to metal-bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl-SAHA and assayed from the observed change in anisotropy. The metal K_D values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, k_off ∼0.0006 s⁻¹, suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 10³ M⁻¹ s⁻¹). Furthermore, monovalent cations (K⁺ or Na⁺) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100-fold for K⁺ and ≥10-fold for Na⁺, suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8.     

Synthesis, Characterization, and Theoretical and Experimental Investigations of Zinc(II)–Amino Acid Complexes as Ecofriendly Plant Growth Promoters and Highly Bioavailable Sources of Zinc

Amino acids (AA) as metal complexing agents have the ability to form relatively stable complexes with zinc (Zn) and thereby increase its availability for plants. In this study, the complexes of Zn(II), [Zn(L–L′)₂] [where L–L′ = monoanion of arginine (Arg), glycine (Gly), glutamine (Gln), histidine (His), and methionine (Met)], were synthesized and characterized by different analytical techniques. The results of elemental analysis support the formation of Zn(II)–AA complexes (ZnAAC) with a 2:1 ligand-to-metal molar ratio. The computational results indicated that the AA ligands coordinated to the Zn(II) ion via their nitrogen and oxygen atoms and support the coordination mode obtained from IR spectroscopy. For the first time, the semiempirical calculations were also performed to investigate the passive uptake of ZnAAC by root cells. The proposed transport pathway indicated that ZnAAC can pass via plant root cell wall pores without any strict hindrances. The efficacy of ZnAAC as a Zn source was evaluated for two lettuce cultivars (Lactuca sativa L., cvs. ‘Lollo Bionda’ and ‘Lollo Rossa’) grown in nutrient solution. The results confirmed the higher efficacy of ZnAAC in supplying Zn for lettuce in comparison with ZnSO₄. The synthesized ZnAAC also had a stimulating effect on root and shoot growth of both lettuce cultivars. According to the results, ZnAAC can be used as ecofriendly plant growth stimulators and sources of Zn to supply plants with readily available Zn.     

Kinetic analysis of the effect of zinc ion on the inorganic pyrophosphatase reaction.

A kinetic study is presented in which the effect of Zn(II) on yeast inorganic pyrophosphatase was quantitatively determined. A dual role model for metal ion effect, previously determined for the Mg(II)-pyrophosphatase system (O. A. Moe and L. G. Butler, 1972, J. Biol. Chem.247, 7308–7315), was applied successfully to the analysis of the kinetics for Zn(II)-pyrophosphatase and Zn(II), Mg(II)-pyrophosphatase systems. The model, assigning an activator role to free Zn(II) ion and a substrate role to the Zn(II)-pyrophosphate complex, gave an excellent fit to the data. Inhibition of the Mg(II)-pyrophosphatase system by Zn(II) was analyzed by a model in which competitive binding of the Mg(II)-pyrophosphate and Zn(II)-pyrophosphate complexes occurred at the enzyme active site, with both complexes undergoing reaction at different rates. Relative maximal velocities and enzymeligand dissociation constants for the Zn(II)-pyrophosphate complex were determined for the cases where the metal ion activator role was fulfilled by Zn(II) and Mg(II), respectively. The maximal velocity parameter showed a dependence on the nature of the activator metal ion, demonstrating that the role of the latter is associated both with the process of substrate binding and with the mechanism of catalysis. Values for all kinetic parameters are reported for an ionic strength of 0.2, pH 7.0, and 25.0 °C.     

Overexpression of barley nicotianamine synthase 1 confers tolerance in the sweet potato to iron deficiency in calcareous soil

Background and aims

Iron (Fe) is an essential micronutrient for all higher organisms. Fe is sparingly available in calcareous soils and Fe deficiency is a major agricultural problem worldwide. Nicotianamine (NA) is a metal chelator involved in metal translocation in plants. Sweet potato is an attractive crop that can grow in poor soil and thus is useful for planting in uncultivated soil. In addition, the sweet potato has recently been suggested as a source of bioethanol. Our aim is to increase NA concentration in sweet potato to ameliorate Fe deficiency.

Method

Sweet potato plants expressing the barley NA synthase 1 (HvNAS1) gene under the control of CaMV 35S promoter were produced by Agrobacterium-mediated transformation.

Results

The transgenic sweet potato exhibited tolerance to low Fe availability when grown in calcareous soil. The level of tolerance to low Fe availability was positively correlated with the HvNAS1 expression level. The NA concentration of the transgenic sweet potato leaves was up to 7.9-fold greater than that of the non-transgenic (NT) plant leaves. Furthermore, the Fe and zinc concentrations were 3- and 2.9-fold greater, respectively, in transgenic sweet potato than in NT plant leaves.

Conclusions

Our results suggest that increasing the NA concentration of sweet potato by overexpression of HvNAS1 could significantly improve agricultural productivity and energy source.

     

The role of heavy-metal ATPases,HMAs, in zinc and cadmium transport in rice

The P_1B-type heavy metal ATPases (HMAs) are diverse in terms of tissue distribution, subcellular localization, and metal specificity. Functional studies of HMAs have shown that these transporters can be divided into two subgroups based on their metal-substrate specificity: a copper (Cu)/silver (Ag) group and a zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) group. Studies on Arabidopsis thaliana and metal hyperaccumulator plants indicate that HMAs play an important role in the translocation or detoxification of Zn and Cd in plants. Rice possesses nine HMA genes, of which OsHMA1–OsHMA3 belong to the Zn/Co/Cd/Pb subgroup. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd, and participates in Zn and Cd transport to developing seeds in rice. OsHMA3 transports Cd and plays a role in the sequestration of Cd into vacuoles in root cells. Modification of the expression of these genes might be an effective approach for reducing the Cd concentration in rice grains.     

Mobilization of Cu and Zn by root exudates of dicotyledonous plants in resin-buffered solutions and in soil

It has been frequently suggested that root exudates play a role in trace metal mobilization and uptake by plants, but there is little in vivo evidence. We studied root exudation of dicotyledonous plants in relation to mobilization and uptake of Cu and Zn in nutrient solutions and in a calcareous soil at varying Cu and Zn supply. Spinach (Spinacia oleracea L.) and tomato (Lycopersicon esculentum L.) were grown on resin-buffered nutrient solutions at varying free ion activities of Cu (pCu 13.0–10.4) and Zn (pZn 10.1–6.6). The Cu and Zn concentrations in the nutrient solution increased with time, except in plant-free controls, indicating that the plant roots released organic ligands that mobilized Cu and Zn from the resin. At same pCu, soluble Cu increased more at low Zn supply, as long as Zn deficiency effects on growth were small. Zinc deficiency was observed in most treatment solutions with pZn ≥ 9.3, but not in nutrient solutions of a smaller volume/plant ratio in which higher Zn concentrations were observed at same pZn. Root exudates of Zn-deficient plants showed higher specific UV absorbance (SUVA, an indicator of aromaticity and metal affinity) than those of non-deficient plants. Measurement of the metal diffusion flux with the DGT technique showed that the Cu and Zn complexes in the nutrient solutions were highly labile. Diffusive transport (through the unstirred layer surrounding the roots) of the free ion only could not explain the observed plant uptake of Cu and of Zn at low Zn²⁺ activity. The Cu and Zn uptake by the plants was well explained if it was assumed that the complexes with root exudates contributed 0.4% (Cu) or 20% (Zn) relative to the free ion. In the soil experiment, metal concentrations and organic C concentrations were larger in the solution of planted soils than in unplanted controls. The SUVA of the soil solution after plant growth was higher for unamended soils, on which the plants were Zn-deficient, than for Zn-amended soils. In conclusion, root exudates of dicotyledonous plants are able to mobilize Cu and Zn, and plants appear to respond to Zn deficiency by exuding root exudates with higher metal affinity.     

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.     

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,characterization and antitumor activity of some metal complexes of 3- and 5-substituted salicylaldehyde o-hydroxybenzoylhydrazones

Complexes of Mn(II), Fe(III), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Pt(II) with 3- and 5-substituted salicylaldehyde o-hydroxybenzoylhydrazones (XSBH, X = H, 3-NO₂, 3-CH₃O, 5-Cl, 5-Br, 5-CH₃ or 5-NO₂) have been prepared and characterized by elemental analysis, conductance measurements, magnetic susceptibilities (from room temperature down to liquid nitrogen temperature) and spectral studies. These studies indicate the following structures: monomeric, high-spin, distorted octahedral for Mn(XSBH)₂; monomeric, high-spin, five-coordinate for Fe(XSBH)SO₄·H₂O; dimeric, high-spin phenoxide bridged, five-coordinate for Fe(XSBH)Cl; dimeric, high-spin five-coordinate for Co(XSBH)Cl·2H₂O; dimeric low-spin, five-coordinate for Ni(XSBH)Cl·2H₂O; dimeric, four-coordinate for Zn(XSBH); and a square-planar structure for M(XSBH)Cl·H₂O (M = Cu(II) or Pt(II).Intermolecular antiferromagnetic exchange interactions are present in Fe(III) complexes, where the exchange parameter (J) is ca. −8.0 cm⁻¹ for these complexes. The Fe(III) complexes exhibit asymmetric quadrupole split doublets in their ⁵⁷Fe Mössbauer spectra. The asymmetry is found to be temperature dependent with relatively symmetrical doublets seen at low temperature. The polycrystalline ESR spectra of Cu(II) complexes are isotropic and indicate a d_x²−y² ground state in square-planar stereo-chemistry. All these metal complexes have been screened for their antitumor activity against the P 388 lymphocytic leukaemia test system in mice and enhanced antitumor activity relative to the free ligand was found but no significant activity at the dosages used.     

Molecular dynamics simulation on the effect of transition metal binding to the N-terminal fragment of amyloid-β

Abstract

We report molecular dynamics simulations of three possible adducts of Fe(II) to the N-terminal 1–16 fragments of the amyloid-β peptide, along with analogous simulations of Cu(II) and Zn(II) adducts. We find that multiple simulations from different starting points reach pseudo-equilibration within 100–300?ns, leading to over 900?ns of equilibrated trajectory data for each system. The specifics of the coordination modes for Fe(II) have only a weak effect on peptide secondary and tertiary structures, and we therefore compare one of these with analogous models of Cu(II) and Zn(II) complexes. All share broadly similar structural features, with mixture of coil, turn and bend in the N-terminal region and helical structure for residues 11–16. Within this overall pattern, subtle effects due to changes in metal are evident: Fe(II) complexes are more compact and are more likely to occupy bridge and ribbon regions of Ramachandran maps, while Cu(II) coordination leads to greater occupancy of the poly-proline region. Analysis of representative clusters in terms of molecular mechanics energy and atoms-in-molecules properties indicates similarity of four-coordinate Cu and Zn complexes, compared to five-coordinate Fe complex that exhibits lower stability and weaker metal–ligand bonding.

Communicated by Ramaswamy H. Sarma