Michael Rutzke

Molecular and Biochemical Characterization of the Selenocysteine Se-Methyltransferase Gene and Se-Methylselenocysteine Synthesis in Broccoli

Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. While different forms of Se vary in their anticarcinogenic efficacy, Se-methylselenocysteine (SeMSC) has been demonstrated to be one of the most effective chemopreventative compounds. Broccoli (Brassica oleracea var. italica) is known for its ability to accumulate high levels of Se with the majority of the selenoamino acids in the form of Se-methylselenocysteine. Therefore, it serves as a good model to study the regulation of SeMSC accumulation in plants. A cDNA encoding selenocysteine Se-methyltransferase, the key enzyme responsible for SeMSC formation, was cloned from broccoli using a homocysteine S-methyltransferase gene probe from Arabidopsis (Arabidopsis thaliana). This clone, designated as BoSMT, was functionally expressed in Escherichia coli, and its identity was confirmed by its substrate specificity in the methylation of selenocysteine. The BoSMT gene represents a single copy sequence in the broccoli genome. Examination of BoSMT gene expression and SeMSC accumulation in response to selenate, selenite, and sulfate treatments showed that the BoSMT transcript and SeMSC synthesis were significantly up-regulated in plants exposed to selenate but were low in plants supplied with selenite. Simultaneous treatment of selenate with selenite significantly reduced SeMSC production. In addition, high levels of sulfate suppressed selenate uptake, resulting in a dramatic reduction of BoSMT mRNA level and SeMSC accumulation. Our results reveal that SeMSC accumulation closely correlated with the BoSMT gene expression and the total Se status in tissues and provide important information for maximizing the SeMSC production in this beneficial vegetable plant.

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.

Genetic and physiological analysis of iron biofortification in maize kernels.

Background Maize is a major cereal crop widely consumed in developing countries, which have a high prevalence of iron (Fe) deficiency anemia. The major cause of Fe deficiency in these countries is inadequate intake of bioavailable Fe, where poverty is a major factor. Therefore, biofortification of maize by increasing Fe concentration and or bioavailability has great potential to alleviate this deficiency. Maize is also a model system for genomic research and thus allows the opportunity for gene discovery. Here we describe an integrated genetic and physiological analysis of Fe nutrition in maize kernels, to identify loci that influence grain Fe concentration and bioavailability. Methodology Quantitative trait locus (QTL) analysis was used to dissect grain Fe concentration (FeGC) and Fe bioavailability (FeGB) from the Intermated B73 × Mo17 (IBM) recombinant inbred (RI) population. FeGC was determined by ion coupled argon plasma emission spectroscopy (ICP). FeGB was determined by an in vitro digestion/Caco-2 cell line bioassay. Conclusions Three modest QTL for FeGC were detected, in spite of high heritability. This suggests that FeGC is controlled by many small QTL, which may make it a challenging trait to improve by marker assisted breeding. Ten QTL for FeGB were identified and explained 54% of the variance observed in samples from a single year/location. Three of the largest FeGB QTL were isolated in sister derived lines and their effect was observed in three subsequent seasons in New York. Single season evaluations were also made at six other sites around North America, suggesting the enhancement of FeGB was not specific to our farm site. FeGB was not correlated with FeGC or phytic acid, suggesting that novel regulators of Fe nutrition are responsible for the differences observed. Our results indicate that iron biofortification of maize grain is achievable using specialized phenotyping tools and conventional plant breeding techniques.

Selenium accumulation in lettuce germplasm

Selenium (Se) is an essential micronutrient for animals and humans. Increasing Se content in food crops offers an effective approach to reduce the widespread selenium deficiency problem in many parts of the world. In this study, we evaluated 30 diverse accessions of lettuce (Lactuca sativa L.) for their capacity to accumulate Se and their responses to different forms of Se in terms of plant growth, nutritional characteristics, and gene expression. Lettuce accessions responded differently to selenate and selenite treatment, and selenate is superior to selenite in inducing total Se accumulation. At least over twofold change in total Se levels between cultivars with high and low Se content was found. Synergistic relationship between Se and sulfur accumulation was observed in nearly all accessions at the selenate dosage applied. The change in shoot biomass varied between lettuce accessions and the forms of Se used. The growth-stimulated effect by selenate and the growth-inhibited effect by selenite were found to be correlated with the alteration of antioxidant enzyme activities. The different ability of lettuce accessions to accumulate Se following selenate treatment appeared to be associated with an altered expression of genes involved in Se/S uptake and assimilation. Our results provide important information for the effects of different forms of Se on plant growth and metabolism. They will also be of help in selecting and developing better cultivars for Se biofortification in lettuce.

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.

Global transcriptional responses of Pseudomonas syringae DC3000 to changes in iron bioavailability in vitro

BackgroundPseudomonas syringae pv tomato DC3000 (DC3000) is a Gram-negative model plant pathogen that is found in a wide variety of environments. To survive in these diverse conditions it must sense and respond to various environmental cues. One micronutrient required for most forms of life is iron. Bioavailable iron has been shown to be an important global regulator for many bacteria where it not only regulates a wide variety of genes involved in general cell physiology but also virulence determinants. In this study we used microarrays to study differential gene regulation in DC3000 in response to changes in levels of cell-associated iron.ResultsDC3000 cultures were grown under highly controlled conditions and analyzed after the addition of iron citrate or sodium citrate to the media. In the cultures supplemented with iron, we found that cell-associated iron increased rapidly while culture densities were not significantly different over 4 hours when compared to cultures with sodium citrate added. Microarray analysis of samples taken from before and after the addition of either sodium citrate or iron citrate identified 386 differentially regulated genes with high statistical confidence. Differentially regulated genes were clustered based on expression patterns observed between comparison of samples taken at different time points and with different supplements. This analysis grouped genes associated with the same regulatory motifs and/or had similar putative or known function.ConclusionThis study shows iron is rapidly taken up from the medium by iron-depleted DC3000 cultures and that bioavailable iron is a global cue for the expression of iron transport, storage, and known virulence factors in DC3000. Furthermore approximately 34% of the differentially regulated genes are associated with one of four regulatory motifs for Fur, PvdS, HrpL, or RpoD.

Toxic and nutrient element concentrations in soft tissues of zebra and quagga mussels from Lakes Erie and Ontario.

Zebra and quagga mussels were collected from Lakes Erie and Ontario in 1997 and the soft mussel tissues were analyzed for Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Se, Sr, V and Zn. No consistent relationships were apparent when comparing element concentrations in soft mussel tissues and mussel type, size range or sampling location. Literature dealing with the absorption of metals by both mussel types is reviewed.

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.

Contaminant and nutrient element levels in soft tissues of zebra and quagga mussels from waters of southern Lake Ontario

Abstract The objective of this study was to compare concentrations of toxic and nutrient elements in soft tissues of two dreissenid mussels, Dreissena polymorpha and a new dreissenid species given the working name of “quagga”, collected from the New York waters of Lake Ontario. In general, elemental concentrations were similar between the two mussel types. The only exceptions were concentrations of Cr, Mo and Zn which were significantly (p

Concentrations of metals in grasses in the vicinity of a municipal refuse incinerator

There is currently much public opposition to the construction of municipal refuse incinerators in the United States owing to health concerns about emitted toxicants. In this study, 19 elements and polychlorinated biphenyls (PCBs) were determined in grasses sampled upwind and downwind from a municipal refuse incinerator which had no emission control devices. Concentrations of Cd, Mo, Hg, Zn, Fe, and Pb were generally highest immediately adjacent to the incinerator. Foliar concentration of the metals, Cd, Mo, Zn, Fe, and Pb was inversely related to the logarithm of the distance downwind from the incinerator. Foliar concentration of Hg decreased linearly with distance downwind. Polychlorinated biphenyls were not detectable in any of the grass samples possibly due to their thermal destruction during incineration or greater dispersion because of their higher vapor pressure. The metal concentration in plants in the vicinity of such incinerators will be affected by the composition of the refuse, incinerator design and stack height, combustion operating conditions, emission control devices, the nature of the plant surface, the water solubility of deposited metal-containing particulates, prevailing weather conditions and root uptake of such metals by plants.