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Publikation

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.
Publikation

Schmidt, H.; Günther, C.; Weber, M.; Spörlein, C.; Loscher, S.; Böttcher, C.; Schobert, R.; Clemens, S.; Metabolome Analysis of Arabidopsis thaliana Roots Identifies a Key Metabolic Pathway for Iron Acquisition PLOS ONE 9, e102444, (2014) DOI: 10.1371/journal.pone.0102444

Fe deficiency compromises both human health and plant productivity. Thus, it is important to understand plant Fe acquisition strategies for the development of crop plants which are more Fe-efficient under Fe-limited conditions, such as alkaline soils, and have higher Fe density in their edible tissues. Root secretion of phenolic compounds has long been hypothesized to be a component of the reduction strategy of Fe acquisition in non-graminaceous plants. We therefore subjected roots of Arabidopsis thaliana plants grown under Fe-replete and Fe-deplete conditions to comprehensive metabolome analysis by gas chromatography-mass spectrometry and ultra-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Scopoletin and other coumarins were found among the metabolites showing the strongest response to two different Fe-limited conditions, the cultivation in Fe-free medium and in medium with an alkaline pH. A coumarin biosynthesis mutant defective in ortho-hydroxylation of cinnamic acids was unable to grow on alkaline soil in the absence of Fe fertilization. Co-cultivation with wild-type plants partially rescued the Fe deficiency phenotype indicating a contribution of extracellular coumarins to Fe solubilization. Indeed, coumarins were detected in root exudates of wild-type plants. Direct infusion mass spectrometry as well as UV/vis spectroscopy indicated that coumarins are acting both as reductants of Fe(III) and as ligands of Fe(II).
Publikation

Schmidt, H.; Böttcher, C.; Trampczynska, A.; Clemens, S.; Use of recombinantly produced 15N3-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry Anal. Bioanal. Chem. 399, 1355-1361, (2011) DOI: 10.1007/s00216-010-4436-7

Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [15N3]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots.
Publikation

Isaure, M.-P.; Sarret, G.; Harada, E.; Choi, Y.-E.; Marcus, M. A.; Fakra, S. C.; Geoffroy, N.; Pairis, S.; Susini, J.; Clemens, S.; Manceau, A.; Calcium promotes cadmium elimination as vaterite grains by tobacco trichomes Geochim. Cosmochim. Acta 74, 5817-5834, (2010) DOI: 10.1016/j.gca.2010.07.011

In tobacco plants, elimination of Zn and Cd via the production of Ca-containing grains at the top of leaf hairs, called trichomes, is a potent detoxification mechanism. This study examines how Cd is incorporated in these biominerals, and how calcium growth supplement modifies their nature. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDX), microfocused X-ray diffraction (μ-XRD), and microfocused X-ray absorption near edge structure (μ-XANES) spectroscopy were used to image the morphology of the grains, identify the crystallized mineral phases, and speciate Cd, respectively. The mineralogy of the grains and chemical form of Cd varied with the amount of Ca. When tobacco plants were grown in a nutrient solution containing 25 μM Cd and low Ca supplement (Ca/Cd = 11 mol ratio), most of the grains were oblong-shaped and low-Cd-substituted calcite. When exposed to the same amount of Cd and high Ca supplement (Ca/Cd = 131 mol ratio), grains were more abundant and diverse in compositions, and in total more Cd was eliminated. Most grains in the high Ca/Cd experiment were round-shaped and composed predominantly of Cd-substituted vaterite, a usually metastable calcium carbonate polymorph, and subordinate calcite. Calcium oxalate and a Ca amorphous phase were detected occasionally in the two treatments, but were devoid of Cd. The biomineralization of cadmium and implications of results for Cd exposure of smokers and phytoremediation are discussed.
Publikation

Harada, E.; Kim, J.-A.; Meyer, A. J.; Hell, R.; Clemens, S.; Choi, Y.-E.; Expression Profiling of Tobacco Leaf Trichomes Identifies Genes for Biotic and Abiotic Stresses Plant Cell Physiol. 51, 1627-1637, (2010) DOI: 10.1093/pcp/pcq118

Nicotiana tabacum (tobacco) plants have short and long glandular trichomes. There is evidence that tobacco trichomes play several roles in the defense against biotic and abiotic stresses. cDNA libraries were constructed from control and cadmium (Cd)-treated leaf trichomes. Almost 2,000 expressed sequence tag (EST) cDNA clones were sequenced to analyze gene expression in control and Cd-treated leaf trichomes. Genes for stress response as well as for primary metabolism scored highly, indicating that the trichome is a biologically active and stress-responsive tissue. Reverse transcription–PCR (RT–PCR) analysis demonstrated that antipathogenic T-phylloplanin-like proteins, glutathione peroxidase and several classes of pathogenesis-related (PR) proteins were expressed specifically or dominantly in trichomes. Cysteine-rich PR proteins, such as non-specific lipid transfer proteins (nsLTPs) and metallocarboxypeptidase inhibitors, are candidates for the sequestration of metals. The expression of osmotin and thaumatin-like proteins was induced by Cd treatment in both leaves and trichomes. Confocal laser scanning microscopy (CLSM) showed that glutathione levels in tip cells of both long and short trichomes were higher than those in other types of leaf cells, indicating the presence of an active sulfur-dependent protective system in trichomes. Our results revealed that the trichome-specific transcriptome approach is a powerful tool to investigate the defensive functions of trichomes against both abiotic and biotic stress. Trichomes are shown to be an enriched source of useful genes for molecular breeding towards stress-tolerant plants.
Publikation

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.
Publikation

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.
Publikation

Böttcher, C.; von Roepenack-Lahaye, E.; Schmidt, J.; Clemens, S.; Scheel, D.; Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray- tandem mass spectrometry J. Mass Spectrom. 44, 466-476, (2009) DOI: 10.1002/jms.1522

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI‐QTOF‐MS and direct infusion ESI‐FTICR‐MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision‐induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl‐ and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed‐phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI‐MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright © 2008 John Wiley & Sons, Ltd.
Publikation

Böttcher, C.; von Roepenack-Lahaye, E.; Schmidt, J.; Schmotz, C.; Neumann, S.; Scheel, D.; Clemens, S.; Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis Plant Physiol. 147, 2107-2120, (2008) DOI: 10.1104/pp.108.117754

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.
Publikation

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.
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