Olena K. Vatamaniuk

Weeds, Worms, and More. Papain's Long-Lost Cousin, Phytochelatin Synthase

This Update is concerned with the mechanism of synthesis of heavy metal-binding thiol peptides, phytochelatins (PCs), by the enzyme PC synthase (EC 2.3.2.15). The bulk of the considerations in this review centers on what has been learned recently of the fundamental mechanics of PC synthesis, the domain organization and phylogenetic distribution of PC synthases, and PC synthase-like enzymes, and what this tells us about the chemistry underlying and the enzyme residues necessary for PC synthesis. It was decided to prepare a review of this type rather than aim at a more comprehensive treatment of heavy metal homeostasis and detoxification in plants for two reasons. The first is that there are already several contemporary reviews dealing with the more global aspects of plant heavy metal physiology. Excellent examples are Cobbett (2000), Clemens (2001), and Cobbett and Goldsbrough (2002). Readers who have not already read these are encouraged to do so. The second reason is that some of the most fascinating and unexpected developments for our understanding in this area of late derive from investigations of the catalytic mechanism and distribution of PC synthases, facets of this field of research that have yet to be reviewed in detail.

Establishing RNA Interference as a Reverse-Genetic Approach for Gene Functional Analysis in Protoplasts

Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Because protoplasts are nongrowing cells, effective RNAi-triggered gene silencing depends not only on a depletion of gene transcripts but also on turnover rates of corresponding polypeptides. Herein, we tested if transient RNAi in protoplasts would result in the depletion of a targeted polypeptide and, because protoplasts have a limited life span, if functional assays of RNAi knockout genes would be feasible in protoplasts. We showed that protoplasts transfection with an in vitro-synthesized dsRNA against Arabidopsis (Arabidopsis thaliana) β-glutamylcysteine synthase (ECS1), a key enzyme in the synthesis of glutathione, resulted in a 95% depletion of ECS1 transcript, a 72% decrease of ECS1 polypeptide, and a 60% drop in glutathione content. These results were comparable with those obtained upon analysis of Arabidopsis seedlings bearing the cad2-1 mutant allele of ECS1. We also improved the procedure for RNAi inactivation of several genes simultaneously. Finally, because we isolated protoplasts from tissues of 14-d-old seedlings instead of 1-month-old mature plants, the described procedure is rapid (as it only takes 20 d from seed planting to functional studies), suitable for analyzing multiple genes in parallel, and independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost- and space-efficient initial screening and selection of genes for subsequent in planta studies.

Tonoplast-localized Abc2 Transporter Mediates Phytochelatin Accumulation in Vacuoles and Confers Cadmium Tolerance

Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates 35S-PC2 uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms.

OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis

This work identifies a physiological substrate and a physiological function of the Arabidopsis oligopeptide transporter, OPT3, in iron (Fe) homeostasis, provides a mechanistic explanation of the role of OPT3 in systemic Fe signaling, and uncovers an aspect of crosstalk between Fe homeostasis and cadmium partitioning. 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.

A γ-Glutamyl Cyclotransferase Protects Arabidopsis Plants from Heavy Metal Toxicity by Recycling Glutamate to Maintain Glutathione Homeostasis

This article identifies and characterizes an Arabidopsis protein, GGCT2;1, which has a cation transport regulator-like (ChaC-like) domain that functions as a γ-glutamyl cyclotransferase. In vivo studies in yeast and Arabidopsis establish that GGCT2;1 recycles Glu as part of the γ-glutamyl cycle and thus maintains GSH homeostasis to counteract heavy metal and metalloids toxicity. Plants detoxify toxic metals through a GSH-dependent pathway. GSH homeostasis is maintained by the γ-glutamyl cycle, which involves GSH synthesis and degradation and the recycling of component amino acids. The enzyme γ-glutamyl cyclotransferase (GGCT) is involved in Glu recycling, but the gene(s) encoding GGCT has not been identified in plants. Here, we report that an Arabidopsis thaliana protein with a cation transport regulator-like domain, hereafter referred to as GGCT2;1, functions as γ-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in Saccharomyces cerevisiae produced phenotypes that were consistent with decreased GSH content attributable to either GSH degradation or the diversion of γ-glutamyl peptides to produce 5-oxoproline (5-OP). 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic (As) via GSH degradation. Recombinant GGCT2;1 converted both GSH and γ-glutamyl Ala to 5-OP in vitro. GGCT2;1 transcripts were upregulated in As-treated Arabidopsis, and ggct2;1 knockout mutants were more tolerant to As and cadmium than the wild type. Overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Under As toxicity, the overexpression lines showed minimal changes in de novo Glu synthesis, while the ggct2;1 mutant increased nitrogen assimilation by severalfold, resulting in a very low As/N ratio in tissue. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress by recycling Glu.

Mutagenic Definition of a Papain-Like Catalytic Triad, Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation, and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase

Phytochelatin (PC) synthases are γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (γ-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes γ-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes γ-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent γ-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary γ-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary γ-Glu-Cys acylation.

Drosophila ABC transporter, DmHMT-1, confers tolerance to cadmium DmHMT-1 and its yeast homolog, SpHMT-1, are not essential for vacuolar phytochelatin sequestration

Half-molecule ATP-binding cassette transporters of the HMT-1 (heavy metal tolerance factor 1) subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans, and Chlamydomonas reinhardtii. Based on studies of S. pombe, it has been proposed that SpHMT-1 transports heavy metal·phytochelatin (PC) complexes into the vacuolysosomal compartment. PCs are glutathione derivatives synthesized by PC synthases (PCS) in plants, fungi, and C. elegans in response to heavy metals. Our previous studies in C. elegans, however, suggested that HMT-1 and PCS-1 do not necessarily act in concert in metal detoxification. To further explore this inconsistency, we have gone on to test whether DmHMT-1, an HMT-1 from a new source, Drosophila, whose genome lacks PCS homologs, functions in heavy metal detoxification. In so doing, we show that heterologously expressed DmHMT-1 suppresses the Cd2+ hypersensitivity of S. pombe hmt-1 mutants and localizes to the vacuolar membrane but does not transport Cd·PC complexes. Crucially, similar analyses of S. pombe hmt-1 mutants extend this finding to show that SpHMT-1 itself either does not transport Cd·PC complexes or is not the principal Cd·PC/apoPC transporter. Consistent with this discovery and with our previous suggestion that HMT-1 and PCS-1 do not operate in a simple linear metal detoxification pathway, we demonstrate that, unlike PCS-deficient cells, which are hypersensitive to several heavy metals, SpHMT-1-deficient cells are hypersensitive to Cd2+, but not to Hg2+ or As3+. These findings significantly change our current understanding of the function of HMT-1 proteins and invoke a PC-independent role for these transporters in Cd2+ detoxification.

COPT6 Is a Plasma Membrane Transporter That Functions in Copper Homeostasis in Arabidopsis and Is a Novel Target of SQUAMOSA Promoter-binding Protein-like 7

Background: Copper uptake is tightly regulated to prevent deficiency while avoiding toxicity. Results: AtCOPT6 localizes to the plasma membrane, is regulated by copper availability, interacts with itself and AtCOPT1, and regulates response to copper limitation and excess. Conclusion: AtCOPT6 is a novel SPL7 target that functions in copper homeostasis in Arabidopsis. Significance: Identification and characterization of copper transporters are crucial for understanding of copper homeostasis. 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 Met106 residue in the TM2 of COPT6 is functionally essential, the conserved Met27 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.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi). Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants). We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 10(6)-10(7) protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

BackgroundTo balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (‘ionome’) of yeast Saccharomyces cerevisiae. Using inductively coupled plasma – mass spectrometry (ICP-MS) we quantify Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S and Zn in 11890 mutant strains, including 4940 haploid and 1127 diploid deletion strains, and 5798 over expression strains.ResultsWe identified 1065 strains with an altered ionome, including 584 haploid and 35 diploid deletion strains, and 446 over expression strains. Disruption of protein metabolism or trafficking has the highest likelihood of causing large ionomic changes, with gene dosage also being important. Gene over expression produced more extreme ionomic changes, but over expression and loss of function phenotypes are generally not related. Ionomic clustering revealed the existence of only a small number of possible ionomic profiles suggesting fitness tradeoffs that constrain the ionome. Clustering also identified important roles for the mitochondria, vacuole and ESCRT pathway in regulation of the ionome. Network analysis identified hub genes such as PMR1 in Mn homeostasis, novel members of ionomic networks such as SMF3 in vacuolar retrieval of Mn, and cross-talk between the mitochondria and the vacuole. All yeast ionomic data can be searched and downloaded at http://www.ionomicshub.org.ConclusionsHere, we demonstrate the power of high-throughput ICP-MS analysis to functionally dissect the ionome on a genome-wide scale. The information this reveals has the potential to benefit both human health and agriculture.