| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 99 === Zn is an essential micronutrient for plants, but it is toxic in excess concentrations. To avoid Zn toxicity, plants have developed Zn homeostasis mechanism to cope with Zn excess in the surrounding soil. The second chapter of my thesis emphasizes the difference in the cross-homeostasis system between Fe and Zn in dealing with excess Zn in the Zn hyperaccumulator Arabidopsis halleri ssp. gemmifera and non-hyperaccumulator Arabidopsis thaliana. Briefly, A. halleri shows low expression of the Fe acquisition and deficiency response-related genes IRT1 and IRT2 as compared to A. thaliana. In A. thaliana, lowering the expression of IRT1 and IRT2 by adding excess Fe to the medium increases Zn tolerance. Excess Zn induces significant Fe deficiency in A. thaliana and reduces Fe accumulation in shoots. By contrast, the accumulation of Fe in shoots of A. halleri was stable under various Zn treatments. Root FRO activity and expression of FIT are low in A. halleri as compared to A. thaliana. Overexpressing a ZIP family member IRT3 in irt1-1, rescues the Fe deficient phenotype and this system also represents A. halleri in which the IRT3 expression is higher. This result demonstrates the role of other ZIP family members in Fe uptake in Arabidopsis. Thus, a fine-tuned Fe homeostasis mechanism in A. halleri maintains optimum Fe level by Zn-regulated ZIP transporters and prevents high Zn uptake through Fe-regulated metal transporters, and in part be responsible for Zn tolerance.
In order to understand the mechanism and various factors involved in Fe-mediated Zn tolerance in A. thaliana, mutant screening was started by using EMS mutant pools. A mutant of zinc tolerance induced by iron 1, designated zir1, was isolated which exhibited a defect in Fe-mediated Zn tolerance and the characterization of this mutant was detailed in the third chapter of this thesis. With map based cloning and genetic complementation, zir1 was identified with a point mutation of glutamic acid residue to lysine on γ-glutamylcysteine synthetase (γ-ECS, GSH1), the enzyme involved in glutathione biosynthesis. The zir1 mutant contains only 10% of the wild-type glutathione level. Blocking glutathione biosynthesis in wild-type plants by buthionine sulfoximine (BSO) a specific inhibitor of GSH1, resulted in the loss of Fe-mediated Zn tolerance. In addition, two other glutathione-deficient mutant alleles of GSH1, pad2-1 and cad2-1, which contain respectively 16% and 33% of wild-type glutathione level, showed comparable decreases in Fe-mediated Zn tolerance. Under conditions of excess Zn and Fe, the recovery of shoot Fe content in pad2-1 and cad2-1 was reduced as compared to the wild-type. Experiments with the phytochelatin (PC)-deficient mutant cad1-3 showed that it possess Fe-mediated Zn tolerance. Furthermore, the induced accumulation of glutathione in response to excess Zn and Fe suggests that glutathione plays a more specific role in Fe-mediated Zn tolerance in Arabidopsis. The fourth chapter of this thesis illustrates the role of glutathione under Fe limited conditions and zir1 was found to be more sensitive to Fe deficient conditions and it grows poorly under alkaline soil. In addition, zir1 showed defect in Fe translocation from root to shoot under Fe limited conditions when compared to wild-type. Analyzing the other glutathione deficient mutant alleles (pad2-1 and cad2-1) also showed more sensitive phenotype under Fe limited conditions. In wild type, the glutathione level was induced under limited Fe supply and blocking glutathione biosynthesis in wild type leads to increased sensitivity to Fe deficiency. Overexpressing GSH1 in wild type leads to increased level of glutathione and showed enhanced tolerance to limited Fe supply. Altogether these data suggest that glutathione is required for the cross-homeostasis between Zn and Fe and glutathione level is associated with tolerance to Fe deficiency and may play a role in Fe mobilization inside the plant.
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