The adverse effect of Cd on growth is apparent from the reduction in root length of the Cd-treated Arabidopsis thaliana roots. The increase of the levels of H2O2 was observed in Cd-treated A. thaliana roots. The lignin biosynthesis related enzymes, POXs and laccases were enhanced during the Cd treatments. The lignin contents slightly increased in Cd-treated A. thaliana roots¡]48 h¡^. The A. thaliana can be tolerant to high concentration of Cd (500 µM), and only part of high levels of H2O2 accumulated in Cd-treated tissues are used by POXs to synthesize the lignin.

The adverse effect of Zn on growth is apparent from the reduction in root length of the Zn-treated Arabidopsis roots. The levels of H2O2 were increased rapidly in Zn-treated Arabidopsis roots. The lignin biosynthesis realated enzymes, peroxidases and laccases were enhanced during the Zn treatments. The lignin contents increased in Zn-treated Arabidopsis roots. Arabidopsis can be tolerant to high concentration of Zn (4 mM), because part of high levels of H2O2 accumulated in Zn-treated tissues are utilized by peroxidases to synthesize the lignin.

Telomerase is the key enzyme synthesizing telomeric DNA in most eukaryotic organisms. In mammals, telomerase expression is abundant in the germline cells but is undetectable in most other differentiated organs. Intensive studies of telomerase have focused on human cancerous cells, where over 90% of all cancerous tissues examined have telomerase activity. In wild-type Arabidopsis, telomerase expression is abundant in reproductive organs and dedifferentiated tissues such as flowers, siliques and calli but barely detectable in vegetative tissues (both rosette and cauline leaves). In this study, a biochemical screen strategy was developed for isolation of telomerase activating mutants in Arabidopsis thaliana. Through screening of Arabidopsis activation-tagged lines by a PCR-based TRAP assay, two tac (for telomerase activator) mutants were isolated. RT-PCR analysis of AtTERT expression revealed that different mechanisms are involved in alternating telomerase activity in tac1 and tac2. We cloned and characterized the TAC1 gene. TAC1 encodes a single zinc finger protein and acts synergistically with auxin to induce telomerase expression without altering cell cycles. Telomere length was unperturbed in the mutant, but other phenotypes, such as altered root development and the ability of cells to grow in culture without exogenous auxin, indicated that TAC1 not only is part of the previously reported link between auxin and telomerase expression, but also potentiates other classic responses to this phytohormone. DNA microarrays were used to analyze the expression profile of the tac1 mutant and revealed that several drought-induced genes were up-regulated 3 to 10 fold in the tac1-1D mutant. RT-PCR analysis further confirmed this up-regulation for five of these genes. Investigation of root growth also indicated that tac1-1D roots were ~20% longer relative to wild-type. Further experiments demonstrated that over-expression of TAC1 does confer drought tolerance, but not salt tolerance. In addition, our preliminary result showed that treatment with a low concentration of IAA could induce drought tolerance in wild-type Arabidopsis. Although plants with constitutive expression of telomerase have no practical utility, the ability of TAC1 to confer drought tolerance could have significant agricultural applications.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains ix, 89 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 82-88).

Thesis (Ph.D.) - University of Glasgow, 2007. / Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Biomedical and Life Sciences (IBLS), University of Glasgow, 2007. Includes bibliographical references. Print version also available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

In Arabidopsis thaliana, a gene family encodes acyl-CoA-binding proteins (ACBPs) conserved at the acyl-CoA-binding domain which facilitates the binding to acyl-CoA esters. These ACBPs, designated ACBP1 to ACBP6, range in size from 10.4 to 73.1 kD. Previous studies have shown that the the overexpression of ACBP1 or ACBP2 in Arabidopsis likely promotes repair of lipid membranes and result in enhanced tolerance to lead and cadmium, respectively. Microarray data (http://bar.utoronto.ca/) revealed that the expression of ACBP1 and ACBP2 is also regulated by other abiotic stresses, such as cold and drought, suggestive of their association with these environmental pressures. The aim of this study is to investigate and better understand the roles of ACBP1 and ACBP2 in different stress responses. It has been previously observed that the expression of both ACBP1 and ACBP4 is lead [Pb(II)]-inducible and recombinant ACBP1 and ACBP4 bind Pb(II) in vitro. In this study, ACBP1 and ACBP4 were overexpressed in Brassica juncea to test if these ACBPs could be extended for application in Pb(II) phytoremediation in transgenic B. juncea. On freezing (-12 to -8 °C) treatment, ACBP1-overexpressing Arabidopsis was freezing sensitive and accumulated more phosphatidic acid (PA), but less phosphatidylcholine (PC), in contrast to acbp1 mutant plants which were freezing tolerant and had reduced PA and elevated PC levels. Such changes in PC and PA were consistent with the expression of the mRNA encoding phospholipase D1 (PLD1), a major enzyme that promotes the hydrolysis of PC to PA. In contrast, the expression of phospholipase D (PLD), which plays a positive role in freezing tolerance, was up-regulated in acbp1 mutant plants and down-regulated in ACBP1-overexpressing plants. Reduced PLD1 expression and decreased hydrolysis of PC to PA may enhance membrane stability in the acbp1 mutant plants. Given that recombinant ACBP1 binds PA and acyl-CoA esters in vitro, the expression of PLD1 and PLD could be regulated by PA or acyl-CoAs maintained by ACBP1, if ACBP1 were to resemble the yeast 10-kD ACBP by its capability to modulate gene expression during stress responses. Interestingly, another membrane-associated ACBP, ACBP2, which shows high (76.9%) conservation in amino acid homology to ACBP1, did not appear to be affected by freezing treatment. Besides freezing stress, ACBP1, as well as ACBP2, have been observed to participate in abscisic acid (ABA) signaling. They both promote ABA signaling in seed germination and seedling development, while only ACBP2 is involved in the drought response. The overexpression of ACBP2 in Arabidopsis up-regulated reactive oxygen species (ROS) production culminating in reduction in stomatal aperture and water loss in guard cells, thereby enhancing drought tolerance. For tests in phytoremediation, B. juncea was selected for overexpression of ACBP1 and ACBP4 because it is fast-growing, has a higher biomass than Arabidopsis, and is known to be a good accumulator of Pb(II). However, results of Pb(II) treatment for two days showed that the overexpression of ACBP1 or ACBP4 in B. juncea did not significantly improve Pb(II) tolerance. Nevertheless, B. juncea overexpressing ACBP1 did accumulate Pb(II) in roots whereas ACBP4-overexpressing B. juncea lines accumulated Pb(II) in both shoots and roots. Given that B. juncea has a larger biomass than Arabidopsis, it is likely that the duration of Pb(II)-incubation tested in this study was not drastic enough for comparison, and the incubation time should be further extended for Pb(II) translocation. In addition, future studies on Arabidopsis should be conducted to better understand the mechanism of ACBP4-mediated Pb(II) accumulation using Arabidopsis acbp4 mutant and ACBP4-overexpressing plants. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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Telomeres are the ends of linear chromosomes tasked with preventing their recognition by the DNA damage machinery and providing a mechanism to solve the end replication problem. The telomeric DNA is mostly double-stranded, but it terminates in a 3’ protrusion termed the G-overhang. Telomeres utilize telomerase, a reverse transcriptase, to elongate the telomere, and thus, solve the end replication problem. Both the double strand region and the G-overhang are bound by specific proteins to facilitate the objectives of the telomere. First discovered in budding yeast, the CST (Cdc13(CTC1)/Stn1/Ten1) complex binds to the G-overhang and is important for both chromosome end protection and telomere replication. Work reported in this dissertation provided the first evidence that CST was present outside of yeast, which led to its subsequent identification in a number of vertebrates. Here I present the identification and characterization of the three components of CST in Arabidopsis thaliana. Similar to yeast, Arabidopsis CST is required for telomere length maintenance, for preventing telomere recombination and chromosome end-to-end fusions. Mutations in the CST complex result in severe genomic instability and stem cells defects. My research also shows that CST and telomerase act synergistically to maintain telomere length. Together these data provide evidence for an essential role for CST in maintaining telomere integrity. Unexpectedly, I discovered that the TEN1 component of CST may have a more complex role than other members of the heterotrimer. The majority of telomere-related functions we can assay using molecular and cytological approaches are shared by CTC1, STN1 and TEN1, though TEN1 has additional roles in maintaining genome stability, modulating telomerase activity and possibly non-telomeric functions in the chloroplast. I also present genetic evidence that TEN1 and STN1 act in the same pathway for the maintenance of telomere length and chromosome end protection. Interestingly, however, disrupting the STN1/TEN1 interaction reveals a separation of STN1 function for chromosome end protection versus telomere length maintenance. Finally, I describe the design and creation of a library of STN1 and TEN1 mutants that will be used to further characterize their functions and their interaction partners. By disrupting such interactions, it will be possible to elucidate the functional significance of these interactions, and thus, provide new insight into how CST functions in Arabidopsis.