Jessica Lye

The A622 gene in Nicotiana glauca (tree tobacco): evidence for a functional role in pyridine alkaloid synthesis.

Nicotiana glauca (Argentinean tree tobacco) is atypical within the genus Nicotiana, accumulating predominantly anabasine rather than nicotine and/or nornicotine as the main component of its leaf pyridine alkaloid fraction. The current study examines the role of the A622 gene from N. glauca (NgA622) in alkaloid production and utilises an RNAi approach to down-regulate gene expression and diminish levels of A622 protein in transgenic tissues. Results indicate that RNAi-mediated reduction in A622 transcript levels markedly reduces the capacity of N. glauca to produce anabasine resulting in plants with scarcely any pyridine alkaloids in leaf tissues, even after damage to apical tissues. In addition, analysis of hairy roots containing the NgA622-RNAi construct shows a substantial reduction in both anabasine and nicotine levels within these tissues, even if stimulated with methyl jasmonate, indicating a role for the A622 enzyme in the synthesis of both alkaloids in roots of N. glauca. Feeding of Nicotinic Acid (NA) to hairy roots of N. glauca containing the NgA622-RNAi construct did not restore capacity for synthesis of anabasine or nicotine. Moreover, treatment of these hairy root lines with NA did not lead to an increase in anatabine levels, unlike controls. Together, these results strongly suggest that A622 is an integral component of the final enzyme complex responsible for biosynthesis of all three pyridine alkaloids in Nicotiana.

Systematic functional characterization of putative zinc transport genes and identification of zinc toxicosis phenotypes in Drosophila melanogaster

SUMMARY The heavy metal zinc is an essential component of the human diet and is incorporated as a structural component in up to 10% of all mammalian proteins. The physiological importance of zinc homeostasis at the cellular level and the molecular mechanisms involved in this process have become topics of increasing interest in recent years. We have performed a systematic functional characterization of the majority of the predicted Drosophila Zip (zinc/iron regulated transporter-related protein) and ZnT genes, using the Gal4-UAS system to carry out both ubiquitous and targeted over-expression and suppression studies for 13 of the 17 putative zinc transport genes identified to date. We found that six of these 13 genes may be essential for fly viability and that three of the remaining seven demonstrate over-expression phenotypes. Our findings reaffirm the previously proposed function of dZnT63C (CG17723: FBgn005432) as an important zinc efflux protein and indicate that the fly homolog of hZip1, dZip42C.1 (CG9428: FBgn0033096), is a strong zinc importer in Drosophila. By combining over-expression of dZip42C.1 with suppression of dZnT63C we were able to produce easily identifiable zinc toxicosis phenotypes, which can be rescued or worsened by modifying dietary zinc content. Our findings show that a genetically based zinc toxicosis situation can be therapeutically treated or exacerbated by modifications to the diet, providing a sensitized background for future, more detailed studies of Zip/ZnT function.

Tissue-specific interplay between copper uptake and efflux in Drosophila

The vinegar fly Drosophila melanogaster is proving to be an excellent system to study the in vivo regulation of the essential metal copper. The Ctr1A/B and DmATP7 copper transport proteins have well-established roles in Drosophila copper uptake and efflux, respectively. Both Ctr1A and DmATP7 are essential genes, whereas Ctr1B mutants are viable but die in excess or depleted copper conditions. Less is known about the tissue-specific requirements for these three genes and how they interact to maintain copper homeostasis in different cell types. Here, we use targeted overexpression and suppression of each gene to examine these questions in vivo. We find that in the epidermal cells that form the adult thoracic and abdominal cuticle, Ctr1A plays a major role in copper uptake, whereas Ctr1B plays only a minor supporting role and DmATP7, as previously shown, is essential for transfer of copper to the trans-Golgi network. We also find that the copper chaperone dSco1 appears necessary for supplying the mitochondria with copper in these tissues. In contrast, in the developing Drosophila eye, DmATP7 appears to be non-essential unless copper levels in these cells are artificially elevated. Again, Ctr1A is the main copper uptake gene in the eye, but when ectopically expressed, Ctr1B has greater phenotypic effects than Ctr1A. Furthermore, Ctr1A and Ctr1B show a dramatic synergistic interaction, indicating their relationship is more complicated than a simply additive one and that they may in fact act cooperatively for optimal copper import.

Detection of Genetically Altered Copper Levels in Drosophila Tissues by Synchrotron X-Ray Fluorescence Microscopy

Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes.

In vivo zinc toxicity phenotypes provide a sensitized background that suggests zinc transport activities for most of the Drosophila Zip and ZnT genes.

Members of the ZIP (SLC39A) and ZnT (SLC30A) families of transmembrane domain proteins are predicted to transport the essential transition metal zinc across membranes, regulating cellular zinc content and distribution via uptake and efflux at the outer plasma and organellar membranes. Twenty-four ZIP and ZnT proteins are encoded in mammalian genomes, raising questions of whether all actually transport zinc, whether several function together in the same tissues/cell types, and how the activity of these transporters is coordinated. To address these questions, we have taken advantage of the ability to manipulate several genes simultaneously in targeted cell types in Drosophila. Previously we reported zinc toxicity phenotypes caused by combining overexpression of a zinc uptake gene, dZip42C.1, with suppression of a zinc efflux gene, dZnT63C. Here we show that these phenotypes can be used as a sensitized in vivo system to detect subtle alterations in zinc transport activity that would be buffered in healthy cells. Using two adult tissues, the fly eye and midline (thorax/abdomen), we find that when overexpressed, most of the 17 DrosophilaZip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity. In most cases, we can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein. Additionally, targeted suppression of each gene by RNA interference reveals several of the fly Zip and ZnT genes are required in the eye, indicating that numerous independent zinc transport genes are acting together in a single tissue.

Compartmentalized zinc deficiency and toxicities caused by ZnT and Zip gene over expression result in specific phenotypes in Drosophila

Movement of zinc ions across cellular membranes is achieved mainly by two families of zinc transport genes encoding multi-transmembrane domain proteins. Members of the Zip family generally transport zinc into the cytosol, either from outside the cell or from the lumen of subcellular organelles such as the endoplasmic reticulum, Golgi, endosomes or storage vacuoles. ZnT proteins move zinc in the opposite direction, resulting in efflux from the cell or uptake into organelles. Zinc homeostasis at both the cellular and systemic level is achieved by the coordinated action of numerous Zip and ZnT proteins, twenty-four in mammals and seventeen in the vinegar fly Drosophila melanogaster. Previously, we have identified a zinc toxicity phenotype in the Drosophila eye, caused by targeted over expression of dZip42C.1 (dZip1) combined with knockdown of dZnT63C (dZnT1). In general, this phenotype was rescued by increased zinc efflux or decreased uptake and was exacerbated by decreased efflux or increased uptake. Now we have identified three additional zinc dyshomeostasis phenotypes caused by over expression of dZnT86D, dZnT86D(eGFP) and dZip71B(FLAG). Genetic and dietary manipulation experiments showed that these three phenotypes all differ both from each other and from our original zinc toxicity phenotype. Based on these data and the approximate subcellular localization of each zinc transport protein, we propose that each phenotype represents a different redistribution of zinc within these cells, resulting in a Golgi zinc toxicity, a Golgi zinc deficiency and a combined Golgi/other organelle zinc toxicity respectively. We are able to group the remaining Drosophila Zip and ZnT genes into several functional categories based on their interaction with the three novel zinc dyshomeostasis phenotypes, allowing the role of each zinc transport protein to be defined in greater detail. This research highlights the differential effects that redistribution of zinc can have within a particular tissue and identifies the Golgi as being particularly sensitive to both excess and insufficient zinc.