Browsing by Subject "cadmium"
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Item Arabidopsis Thaliana CARBOXYL-TERMINAL DOMAIN PHOSPHATASE-Like1 (CPL1) Mediates Responses to Iron Deficiency and Cadmium Toxicity(2014-04-24) Aksoy, EmreThe expression of genes that control iron (Fe) uptake and distribution (i.e., Fe utilization- related genes) is under a strict regulation. Fe deficiency strongly induces Fe utilization- related gene expression; however, little is known about the mechanisms that regulate this response in plants. In this dissertation, a RNA metabolism factor, RNA POLYMERASE II CTD-PHOSPHATASE-LIKE1 (CPL1) was shown to localize to the root stele, and to be involved in the regulation of Fe deficiency responses in Arabidopsis thaliana. An analysis of multiple cpl1 alleles established that cpl1 mutations enhanced transcriptional responses of Fe utilization-related genes, e.g. IRON-REGULATED TRANSPORTER1 (IRT1), to low Fe availability. In addition to the lower Fe content in the roots, but higher Fe content in the shoots of cpl1-2 plants, the root growth of cpl1-2 showed improved tolerance to Fe deficiency. Genetic data indicated that cpl1-2 likely activates Fe deficiency responses upstream of both FE?DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)- dependent and -independent signaling pathways. Interestingly, various osmotic stress/ABA-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. Unlike Fe, accumulation of the heavy-metal cadmium (Cd) in plants is toxic and it is absorbed by the roots due to the low selectivity of metal transporters such as AtIRT1. In this dissertation, CPL1 was also shown to regulate the transcriptional responses to Cd toxicity. cpl1-2 showed higher tolerance to the Cd toxicity by enhancing the root-to-shoot translocation of Cd by an unknown mechanism. A knowledge-based screening resulted in identification of a putative metal transporter, OLIGOPEPTIDE TRANSPORTER (OPT), which was highly induced in cpl1-2 upon exposure to Cd. OPT was localized to the plastids, indicating a role of plastids in Cd transport and accumulation. The root growth of opt mutants showed higher tolerance to the Cd toxicity, and the mutants accumulated less Cd, Fe and Zn, indicating the involvement of OPT in the transport of these metals. This presented dissertation suggests that 1) CPL1 functions as a negative regulator of the Fe deficiency signaling at the crosstalk with a branch of the osmotic stress/ABA signaling pathway, and 2) CPL1 regulates the Cd distribution in plants by repressing the expression of OPT.Item The Pro-Antioxidant Role of Zinc Supplementation in Cadmium-Treated Choroid Plexus(2014-05-06) Francis Stuart, Samantha D.Cadmium (Cd) is a toxic heavy metal with no known physiological function in higher order animals. Previous studies in primary cultures of neonatal rat choroid plexus (CP) epithelial cells indicated Cd induced oxidative stress and stimulated apical choline transport, and suggested zinc (Zn) supplementation might abate both oxidative stress and modulation of transport. The objective of this thesis was to elucidate how Zn, a nutritive mineral normally accumulated by CP, attenuated oxidative stress. I hypothesize that Zn, which can function as a pro-antioxidant, abates Cd-induced oxidative stress either by induction of metallothionein-1 (MT-1) or enhancement of glutathione (GSH) biochemistry. Thus, in primary cultures of neonatal rat CP epithelial cells, I characterized the effects of sub-micromolar Cd and efficacy of Zn supplementation to attenuate Cd-induced cellular and oxidative stress without or with manipulation of GSH synthesis. To characterize the Cd-induced stress response, CP epithelial cells were treated with 0 or 500 nM CdCl_(2) in serum-free medium (SFM) for 12 h; samples were collected at 3, 6, 9, and 12 h. Induction of heme oxygenase-1 (HO-1), heat-shock protein 70 (HSP70), and metallothionein-1 (MT-1) in Cd-treated cells was compared to time-matched controls by immunoblot and qRT-PCR analyses. Cd induced the catalytic and modifier subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis. To elucidate the effects Zn supplementation in Cd-treated cells depleted of GSH, cells were supplemented for 48 h with 0 or 25 ?M ZnCl_(2) alone or with 100 ?M buthionine sulfoximine (BSO), an inhibitor of GCL, before treatment with 0 or 500 nM CdCl_(2) ? 100 ?M BSO ? 10?M ZnCl_(2) in SFM for 12 h. By luminescence assay, intracellular GSH and oxidized glutathione (GSSG) concentrations were measured. Cd increased intracellular GSH and GSSG, but markedly decreased GSH:GSSG ratio. Inhibition of GSH synthesis exacerbated Cd-induced stress. However, Zn supplementation attenuated the stress response irrespective of BSO treatment, as per decreased induction of HSP70. These data indicate that CP adapts to low-dose Cd by up-regulation of stress proteins and GSH synthesis. Zinc supplementation also may attenuate Cd-induced cellular and oxidative stress, but cytoprotection is independent of GSH status.