Phytochelatins, a class of posttranslationally synthesized peptides, play a pivotal role in heavy metal, primarily Cd 2؉ , tolerance in plants and fungi by chelating these substances and decreasing their free concentrations. Derived from glutathione and related thiols by the action of ␥-glutamylcysteine dipeptidyl transpeptidases (phytochelatin synthases; EC 2.3.2.15), phytochelatins consist of repeating units of ␥-glutamylcysteine followed by a C-terminal Gly, Ser, or ␤-Ala residue [poly-(␥-Glu-Cys) n -Xaa]. Here we report the suppression cloning of a cDNA (AtPCS1) from Arabidopsis thaliana encoding a 55-kDa soluble protein that enhances heavy-metal tolerance and elicits Cd 2؉ -activated phytochelatin accumulation when expressed in Saccharomyces cerevisiae. On the basis of these properties and the sufficiency of immunoaffinity-purified epitopetagged AtPCS1 polypeptide for high rates of Cd 2؉ -activated phytochelatin synthesis from glutathione in vitro, AtPCS1 is concluded to encode the enzyme phytochelatin synthase.Essential heavy metals, such as Cu and Zn, are required as cofactors in redox reactions and ligand interactions as well as for charge stabilization, charge shielding, and water ionization during biocatalysis (1). However, both essential and nonessential heavy metals can pose an acute problem for organisms. Supraoptimal concentrations of essential heavy metals and micromolar concentrations of nonessential heavy metals, such as As, Cd, Hg, and Pb, displace endogenous metal cofactors, heavy or otherwise, from their cellular binding sites, undergo aberrant reactions with the thiol groups of proteins and coenzymes, and promote the formation of active oxygen species (2).Three classes of peptides, glutathione (GSH), metallothioneins (MTs), and phytochelatins (PCs), have been implicated in heavy-metal homeostasis in plants. The thiol peptide, GSH (␥-Glu-Cys-Gly), and in some species its variant homoglutathione (h-GSH, ␥-Glu-Cys-␤-Ala), is considered to influence the form and toxicity of heavy metals such as As, Cd, Cu, Hg, and Zn, in several ways. These include direct metal binding (3), promotion of the transfer of metals to other ligands, such as MTs and PCs (4), provision of reducing equivalents for the generation of metal oxidation states more amenable to binding by MTs and possibly PCs (4), removal of the active oxygen species formed as a result of exposure of cells to heavy metals (5), and͞or the formation of transport-active metal complexes (6). MTs, small (4-to 8-kDa) Cys-rich metal-binding peptides containing multiple Cys-Xaa-Cys motifs, confer tolerance to a broad range of metals in mammals (7) but appear to be involved primarily in Cu homeostasis in plants (8). Arabidopsis MT1 and MT2 confer tolerance to high levels of Cu 2ϩ but only low levels of Cd 2ϩ when heterologously expressed in MT-deficient cup1⌬ mutants of Saccharomyces cerevisiae (8), MT expression in Arabidopsis seedlings is strongly induced by Cu 2ϩ but not by Cd 2ϩ

Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.

The dependence of phytochelatin synthase (␥-glutamylcysteine dipeptidyltranspeptidase (PCS), EC 2.3.2.15) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (At-PCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd 2؉ -and Zn 2؉-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd⅐GS 2 or Zn⅐GS 2 ) and free glutathione act as ␥-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both ␥-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.

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A deficiency of the micronutrient copper (Cu) leads to infertility and grain/seed yield reduction in plants. How Cu affects fertility, which reproductive structures require Cu, and which transcriptional networks coordinate Cu delivery to reproductive organs is poorly understood. Using RNA-seq analysis, we showed that the expression of a gene encoding a novel transcription factor, CITF1 (Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR1), was strongly upregulated in Arabidopsis thaliana flowers subjected to Cu deficiency. We demonstrated that CITF1 regulates Cu uptake into roots and delivery to flowers and is required for normal plant growth under Cu deficiency. CITF1 acts together with a master regulator of copper homeostasis, SPL7 (SQUAMOSA PROMOTER BINDING PROTEIN LIKE7), and the function of both is required for Cu delivery to anthers and pollen fertility. We also found that Cu deficiency upregulates the expression of jasmonic acid (JA) biosynthetic genes in flowers and increases endogenous JA accumulation in leaves. These effects are controlled in part by CITF1 and SPL7. Finally, we show that JA regulates CITF1 expression and that the JA biosynthetic mutant lacking the CITF1-and SPL7-regulated genes, LOX3 and LOX4, is sensitive to Cu deficiency. Together, our data show that CITF1 and SPL7 regulate Cu uptake and delivery to anthers, thereby influencing fertility, and highlight the relationship between Cu homeostasis, CITF1, SPL7, and the JA metabolic pathway.

Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma membrane and mediates copper accumulation when expressed in the Saccharomyces cerevisiae copper uptake mutant. Although the primary sequence of COPT6 contains the family conserved domains, including methionine-rich motifs in the extracellular N-terminal domain and a second transmembrane helix (TM2), it is different from the founding family member, S. cerevisiae Ctr1p. This conclusion was based on the finding that although the positionally conserved Met(106) residue in the TM2 of COPT6 is functionally essential, the conserved Met(27) in the N-terminal domain is not. Structure-function studies revealed that the N-terminal domain is dispensable for COPT6 function in copper-replete conditions but is important under copper-limiting conditions. In addition, COPT6 interacts with itself and with its homolog, COPT1, unlike Ctr1p, which interacts only with itself. Analyses of the expression pattern showed that although COPT6 is expressed in different cell types of different plant organs, the bulk of its expression is located in the vasculature. We also show that COPT6 expression is regulated by copper availability that, in part, is controlled by a master regulator of copper homeostasis, SPL7. Finally, studies using the A. thaliana copt6-1 mutant and plants overexpressing COPT6 revealed its essential role during copper limitation and excess.

Phytochelatins (PCs), (␥-Glu-Cys) n Gly polymers that were formerly considered to be restricted to plants and some fungal systems, are now known to play a critical role in heavy metal (notably Cd 2؉ ) detoxification in Caenorhabditis elegans. In view of the functional equivalence of the gene encoding C. elegans PC synthase 1, ce-pcs-1, to its homologs from plant and fungal sources, we have gone on to explore processes downstream of PC fabrication in this organism. Here we describe the identification of a half-molecule ATP-binding cassette transporter, CeHMT-1, from C. elegans with an equivalent topology to that of the putative PC transporter SpHMT-1 from Schizosaccharomyces pombe. At one level, Ce-HMT-1 satisfies the requirements of a Cd . These results and those from our previous investigations of the requirement for PC synthase for heavy metal tolerance in C. elegans demonstrate PC-dependent, HMT-1-mediated heavy metal detoxification not only in S. pombe but also in some invertebrates while at the same time indicating that the action of Ce-HMT-1 does not depend exclusively on PC synthesis.The toxicity of supraoptimal levels of the essential heavy metals copper and zinc and of trace or higher levels of the nonessential heavy metals cadmium, arsenic, mercury, and lead is thought to result from the displacement of endogenous co-factors from their cellular binding sites, thiol-capping of essential proteins and peptides, and promotion of the formation of reactive oxygen species (1). In humans, the repercussions of heavy metal exposure can range from acute poisoning to progressive kidney, liver, and lung dysfunction and, in some instances, cancer. Although its true clinical and epidemiological significance remains to be determined, chronic exposure to heavy metals is often associated with muscular and neurological degenerative conditions reminiscent of muscular dystrophy, multiple sclerosis, Alzheimer disease, and Parkinson disease (2-4).Three classes of heavy metal-binding peptides are known to participate in the homeostasis and detoxification of heavy metals in most animals. These are the ubiquitous thiol tripeptide glutathione (GSH), a family of small (4 -8 kDa) cysteine-rich proteins termed metallothioneins, and several higher molecular mass albeit sequence-unrelated metal-binding proteins. In all of the plants studied and in some fungi (as exemplified by Schizosaccharomyces pombe and Candida glabrata) and some animals (as exemplified by the model nematode Caenorhabditis elegans), a third class of cysteine-rich peptides termed phytochelatins (PCs) 1 has also been shown to be involved in heavy metal detoxification.PCs, which constitute a family of short-chain heavy metalbinding peptides with the general structure (␥-EC) n Xaa, where n ϭ 2-11, are derived from GSH and related thiols in a ␥-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (EC 2.3.2.15) (1,5,6). It is now almost 15 years since the partial purification of the enzyme capable of catalyzing PC synthesis (5), yet it is onl...

SUMMARYThe bone morphogenetic protein (BMP) signaling pathway regulates multiple developmental and homeostatic processes. Mutations in the pathway can cause a variety of somatic and hereditary disorders in humans. Multiple levels of regulation, including extracellular regulation, ensure proper spatiotemporal control of BMP signaling in the right cellular context. We have identified a modulator of the BMP-like Sma/Mab pathway in C. elegans called DRAG-1. DRAG-1 is the sole member of the repulsive guidance molecule (RGM) family of proteins in C. elegans, and is crucial in regulating body size and mesoderm development. Using a combination of molecular genetic and biochemical analyses, we demonstrate that DRAG-1 is a membraneassociated protein that functions at the ligand-receptor level to modulate the Sma/Mab pathway in a cell-type-specific manner. We further show that DRAG-1 positively modulates this BMP-like pathway by using a novel Sma/Mab-responsive reporter. Our work provides a direct link between RGM proteins and BMP signaling in vivo and a simple and genetically tractable system for mechanistic studies of RGM protein regulation of BMP pathways.