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Publications - Stress and Develop Biology

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Publications

Kühnlenz, T.; Westphal, L.; Schmidt, H.; Scheel, D.; Clemens, S.; Expression of Caenorhabditis elegans PCS in the AtPCS1-deficient Arabidopsis thaliana cad1-3 mutant separates the metal tolerance and non-host resistance functions of phytochelatin synthases Plant Cell Environ. 38, 2239-2247, (2015) DOI: 10.1111/pce.12534

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.
Publications

Tennstedt, P.; Peisker, D.; Böttcher, C.; Trampczynska, A.; Clemens, S.; Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc Plant Physiol. 149, 938-948, (2009) DOI: 10.1104/pp.108.127472

The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.
Publications

Lin, Y.-F.; Liang, H.-M.; Yang, S.-Y.; Boch, A.; Clemens, S.; Chen, C.-C.; Wu, J.-F.; Huang, J.-L.; Yeh, K.-C.; Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/iron transporter New Phytol. 182, 392-404, (2009) DOI: 10.1111/j.1469-8137.2009.02766.x

ZIP transporters (ZRT, IRT‐like proteins) are involved in the transport of iron (Fe), zinc (Zn) and other divalent metal cations. The expression of IRT3, a ZIP transporter, is higher in the Zn/cadmium (Cd) hyperaccumulator Arabidopsis halleri than is that of its ortholog in Arabidopsis thaliana , which implies a positive association of its expression with Zn accumulation in A. halleri. IRT3 genes from both A. halleri and A. thaliana functionally complemented the Zn uptake mutant Spzrt1 in Schizosaccharomyces pombe ; and Zn uptake double mutant zrt1zrt2 , Fe‐uptake mutant fet3fet4 and conferred Zn and Fe uptake activity in Saccharomyces cerevisiae . By contrast, the manganese (Mn) uptake mutant smf1 phenotypes were not rescued. Insufficient Cd uptake for toxicity was found.Expression of IRT3‐green fluorescent protein (GFP) fusion proteins in Arabidopsis root protoplasts indicated localization of both IRT3 proteins in the plasma membrane.Overexpressing AtIRT3 in A. thaliana led to increased accumulation of Zn in the shoot and Fe in the root of transgenic lines. Therefore, IRT3 functions as a Zn and Fe‐uptake transporter in Arabidopsis.
Publications

Boch, A.; Trampczynska, A.; Simm, C.; Taudte, N.; Krämer, U.; Clemens, S.; Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 in Schizosaccharomyces pombe reveals the delicacy of cellular zinc balance FEMS Yeast Res. 8, 883-896, (2008) DOI: 10.1111/j.1567-1364.2008.00414.x

Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn2+ ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae. We identified the first Zn2+-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1Δ cells were unable to grow at low Zn2+ and showed reduced65Zn2+ uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd2+ ions inhibited uptake but Fe2+ or Mn2+ did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn2+ concentrations. Optimal growth is restricted to a narrow range of Zn2+ concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.
Publications

Clemens, S.; Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants Biochimie 88, 1707-1719, (2006) DOI: 10.1016/j.biochi.2006.07.003

Over the past 200 years emissions of toxic heavy metals have risen tremendously and significantly exceed those from natural sources for practically all metals. Uptake and accumulation by crop plants represents the main entry pathway for potentially health-threatening toxic metals into human and animal food. Of major concern are the metalloids arsenic (As) and selenium (Se), and the metals cadmium (Cd), mercury (Hg), and lead (Pb). This review discusses the molecular mechanisms of toxic metal accumulation in plants and algae, the responses to metal exposure, as well as our understanding of metal tolerance and its evolution. The main emphasis will be on cadmium, which is by far the most widely studied of the non-essential toxic metals/metalloids. Entry via Zn2+, Fe2+, and Ca2+ transporters is the molecular basis of Cd2+ uptake into plant cells. Much less is known about the partitioning of non-essential metals and about the genes underlying the enormous diversity among plants with respect to Cd accumulation in different tissues. Numerous studies have described symptoms and responses of plants upon toxic metal exposure. Mysterious are primary targets of toxicity, the degree of specificity of responses, the sensing and the signaling events that lead to transcriptional activation. All plants apparently possess a basal tolerance of toxic non-essential metals. For Cd and As, this is largely dependent on the phytochelatin pathway. Not understood is the molecular biology of Cd hypertolerance in certain plant species such as the metallophytes Arabidopsis halleri or Thlaspi caerulescens.
Publications

Clemens, S.; Evolution and function of phytochelatin synthases J. Plant Physiol. 163, 319-332, (2006) DOI: 10.1016/j.jplph.2005.11.010

Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.
Publications

Weber, M.; Trampczynska, A.; Clemens, S.; Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri Plant Cell Environ. 29, 950-963, (2006) DOI: 10.1111/j.1365-3040.2005.01479.x

Toxic effects of both essential and non‐essential heavy metals are well documented in plants. Very little is known, however, about their modes of toxicity, about tolerance mechanisms and the signalling cascades involved in mediating transcriptional responses to toxic metal excess. We analysed transcriptome changes upon Cd2+ and Cu2+ exposure in roots of Arabidopsis thaliana and the Cd2+‐hypertolerant metallophyte Arabidopsis halleri . Particularly, three categories of genes were identified with the help of this comparative approach: (1) common responses, which might indicate stable and functionally relevant changes conserved across plant species; (2) metallophyte‐specific responses as well as transcripts differentially regulated between the two species, representing candidate genes for Cd2+ hypertolerance; and (3) those specifically responsive to Cd2+ and therefore indicative of toxicity mechanisms or potentially involved in signalling cascades. Our data define, for instance, Arabidopsis core responses to Cd2+ and Cu2+. In addition, they suggest that Cd2+ exposure very rapidly results in apparent Zn deficiency, and they show the existence of highly specific Cd2+ responses and distinct signalling cascades. Array results were independently confirmed by real‐time quantitative PCR, thereby further validating cross‐species transcriptome analysis with oligonucleotide microarrays.
Publications

Trampczynska, A.; Böttcher, C.; Clemens, S.; The transition metal chelator nicotianamine is synthesized by filamentous fungi FEBS Lett. 580, 3173-3178, (2006) DOI: 10.1016/j.febslet.2006.04.073

Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+‐hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray‐ionization‐quadrupole‐time‐of‐flight mass spectrometry we prove the formation of nicotianamine in N. crassa . Transcript level is strongly upregulated under Zn deficiency as shown by real‐time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions.
Publications

Roth, U.; von Roepenack-Lahaye, E.; Clemens, S.; Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+ J. Exp. Bot. 57, 4003-4013, (2006) DOI: 10.1093/jxb/erl170

Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure—which directly influence accumulation rates—are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 μM Cd2+ for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average ∼1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd2+ treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur.
Publications

Grzam, A.; Tennstedt, P.; Clemens, S.; Hell, R.; Meyer, A. J.; Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase FEBS Lett. 580, 6384-6390, (2006) DOI: 10.1016/j.febslet.2006.10.050

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione‐bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the γ‐glutamylcysteine‐bimane intermediate (γ‐EC‐B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine‐bimane without intermediate. Only co‐infiltration of monochlorobimane with Cd2+ and Cu2+ increased γ‐EC‐B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null‐mutants. In the absence of elevated heavy metal concentrations glutathione‐conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate‐limiting.
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