Browsing by Subject "Arabidopsis thaliana"
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Item Dissection of the telomere complex CST in Arabidopsis thaliana(2013-07-26) Leehy, KatherineTelomeres 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.Item Distribution and expression of apyrases in pea and Arabidopsis(2003) Sun, Yu, doctor of computer sciences; Roux, Stanley J.This dissertation describes the results of experiments aimed at defining the tissue distribution, regulated expression and nuclear function of apyases in both Pisum sativum and Arabidopsis thaliana. As an approach to tissue-specific localizations in Arabidopsis, promoter regions including 5’UTR of two Arabidopsis apyrases, Atapy1 and Atapy2, were linked with GUS and transformed into Arabidopsis thaliana. As evaluated by GUS staining, the expression patterns of the two apyrases were almost identical. The highest expression level for both apyrases was found in mature pollen, the stigma surface, root cap and columella cells, the abscission zone and the top part of stipules. Both apyrases were also expressed highly in selected cell types of roots other than root tip. However, expression in hypocotyls and in flowering stems was not detectable for either apyrase in light grown seedlings. In etiolated seedlings, both apyrases are expressed in the upper region of the hypocotyl. Semi-quantitative RT-PCR measurements showed that both apyrases are upregulated by blue light and red light. Quantitative GUS assay and RT-PCR also shows that Atapy1 and Atapy2 are differently regulated by physical injury. Possible functions of AtAPYs in secretion, wound responses and in phytochromemediated light signal transduction are proposed. Transgenic plants that express AtAPY1-GFP and the antisense of Atapy1 were also made. The AtAPY1-GFP hybrid protein was found to exist as dot-like structures primarily in the cytosol. These plants showed no visible phenotype differences to wild-type plants, in conformity to the observations that plants knocked out in Atapy1 had no apparent phenotypic differences from wild-type plants. In the nuclei of etiolated pea seedlings, expression of the apyrase protein is regulated by red light but not white light. The nuclear apyrase was found to exist in a large (MW ~ 400Kd-800Kd) protein complex bound to the nuclear matrix. Specific molecular mass proteins were found to associate with apyrase when it was separated on molecular sieve chromatography and immunoprecipitated with highly specific polyclonal antibodies. In Arabidopsis, attempts to use AtAPY1 as bait in the yeast two-hybrid system to search for its binding protein were not successful.Item Evolutionary genetics and ecology of water use efficiency ([delta]¹³C) in Ipomopsis agregata and Arabidopsis thaliana(2011-12) Kenney, Amanda Marie; Juenger, Thomas; Bolnick, Daniel I.; Linder, C. Randal; Roux, Stanley J.; McKay, John K.My dissertation research investigates the genetic architecture and evolutionary significance of physiological variation in two wildflower species, Ipomopsis aggregata and Arabidopsis thaliana. In particular, my work focuses on water use efficiency (WUE), a critical physiological trait that dictates plant growth and performance in resource-limited environments. I used a combination of quantitative trait loci (QTL) mapping, field selection experiments, and classic quantitative genetics to investigate 1) the genetic architecture of water use efficiency and flowering time, 2) patterns of natural selection on water use efficiency, flowering time, and other ecological traits in I. aggregata, and 3) additive genetic variation, genetic correlations, and selection on water use efficiency, flowering time, and plasticity to drought in Arabidopsis thaliana. Using an Ipomopsis aggregata genetic mapping population, I identified four QTL underlying WUE, three QTL-QTL epistatic interactions, and evidence for a possible QTL x cytoplasmic interaction affecting WUE. I found a similar genetic architecture underlying flowering time, with four main effect QTLs that all adjacently localized to the same linkage groups as WUE, and three QTL-QTL epistatic interactions, which occur between the same chromosome pairs as the WUE interactions. The combined main and interactive effects explain 35% and 40% of the phenotypic variation in WUE and flowering time, respectively. The adjacent localization suggests a possible role for the evolution of co-inheritance or, if the true QTL positions actually overlap, a possible role for pleiotropy underlying the phenotypic correlation between WUE and flowering time. Additionally, these results suggest epistasis is a significant factor affecting phenotypic variation in nature. In a reciprocal transplant and water addition experiment, I demonstrated variable natural selection on WUE, flowering time, and nectar production in I. aggregata across elevation/habitat and differential water availability. At low elevation in the water addition treatment, natural selection favors early flowering and greater nectar sugar concentration, while dry conditions favor high WUE and early flowering time. At high elevation, where the growing season is shorter and drier, selection favors early flowering regardless of water addition. These results suggest natural selection on ecophysiological and floral traits varies with resource availability (e.g. water availability and pollinator visitation). Using data from a glasshouse experiment involving a global panel of accessions of Arabidopsis thaliana, I demonstrated strong positive genetic correlation between WUE and flowering time, as well as selection for low WUE and early flowering under experimental season-ending drought. Finally, I found significant genetic variation in plasticity as well as selection favoring greater WUE plasticity under drought, indicating plasticity to drought is adaptive in A. thaliana.Item Extracellular ATP signaling: induction of superoxide accumulation and possible regulation by ectoapyrases in Arabidopsis thaliana(2004) Song, Charlotte Jarlen; Roux, Stanley J.Plants are constantly assaulted with abiotic and biotic stresses. They have developed mechanisms to transduce those stresses into adaptive physiological responses, including the production of reactive oxygen species as an intermediate signal, a mechanism they share in common with animal responses to stress. In animals, extracellular ATP (xATP) is a signal that induces the production of reactive oxygen species. In this dissertation, we document that xATP can serve as a signal also in plants by inducing superoxide production via NADPH oxidases. We also characterize intermediate elements in the ATP signaling pathway, downstream gene expression changes and the possible regulation of this signal by apyrases in Arabidopsis thaliana. ATP- treated Arabidopsis leaves had increased superoxide accumulation. Inhibitors of the P2 receptors that initiate xATP responses in animals were able to block ATP-induced superoxide production in Arabidopsis. Mutants missing NADPH oxidase subunits did not show ATP-induced superoxide accumulation, indicating that NADPH oxidase activity is responsible for the ATP-induced superoxide production. A cation channel blocker, a calcium chelator, and a calmodulin antagonist also blocked this ATP response, implicating increases in [Ca2+]cyt and the activation of calmodulin as intermediate signaling steps between xATP and superoxide production. Genes that are induced by various stresses were up-regulated by xATP, including genes involved in the biosynthesis of jasmonates and ethylene. Ectoapyrases are enzymes that hydrolyze extracellular nucleotides. Arabidopsis apyrases were assessed as possible regulators of the xATP signal, a role they could carry out directly by quenching the signal and indirectly by increasing the AMP product that can readily be converted to adenosine, a negative feedback suppressor of ATP effects in animals, although this has not been demonstrated in plants to date. Adenosine decreased ATP-induced superoxide production, and three independent lines overexpressing the apyrase gene, Atapy2 (Arabidopsis thaliana apyrase 2) had reduced superoxide production in response to ATP treatment. The level of two mRNAs encoding apyrases increased in response to wounding, but only Atapy2 had increased gene expression when treated with oligalacturonic acid (OGA). These differences suggest differential regulation of these similar apyrases. This dissertation provides evidence for a novel signal transduction pathway in Arabidopsis leading to the induction of superoxide production and the possible regulation of this pathway by apyrases.Item Genetic analyses of adaptive evolution in seed oil composition in the model plant Arabidopsis thaliana : a quantitative genetic approach(2010-08) Sanyal, Anushree; Linder, C. Randal; Juenger, Thomas E.; Lloyd, Alan M.; Simpson, Beryl B.; Mueller, Ulrich G.Natural variation in the relative proportions of saturated and unsaturated fatty acids in seed oils of plants is enormous when considered across a broad taxonomic range of oil seeds. It has been shown that this variation follows a latitudinal cline where the proportions of unsaturated fatty acids increases with increasing latitude as the unsaturated fatty acids in seeds provide energy at a faster rate to germinating seeds at higher latitudes. This variation which follows a latitudinal cline suggests that there may be an adaptive role for this variation. We tested this hypothesis in Arabidopsis thaliana which followed the same trend seen in Helianthus and other angiosperms. In order to understand the underlying genetics of the regulation of the relative proportions of fatty acids and their role in plant evolution, we mapped quantitative trait loci (QTLs) and candidate genes. Here we identified 67 major QTLs responsible for fatty acid synthesis in A. thaliana in Ler-0 x Sha, Ler-0 x Col-4, Ler-2 x Cvi and Ler-0 x No-0 RIL populations. Eight candidate genes were identified based on what is known about seed oil biosynthesis in A. thaliana. Six of the candidate genes collocated to most of the major QTLs. In order to demonstrate that a particular allelic variant is indeed causally related to the phenotype, we investigated DNA polymorphisms in the parental and the RIL line alleles of the collocating candidate genes. Single nucleotide polymorphisms (SNPs) were identified in the collocating candidate genes to study the correlation between the sequence variants and the particular phenotype. We identified 232 SNPs with 77 in the putative regulatory regions upstream of the 5’UTR, 61 in the introns, 18 in the 5’UTR regions, 2 in the 3’UTR regions, and 45 occurring in the exons with 10 non-synonymous substitutions affecting the amino acid residues. We also detected 44 insertions/deletions in the coding, non-coding, 5’UTR, 3’UTR and the regulatory regions. Sequence variation in the fatty acid genes due to SNPs and insertions/deletions should be valuable in tests of association to investigate how the relative proportions of saturated and unsaturated fatty acids are regulated in wild plants and what role they have played in plant evolution and also in breeding oil seed crops that are healthier or have two types of fatty acids in proportions appropriate for different uses.Item Genetic analyses of natural variation in the model plant Arabidopsis thaliana: neutral marker, quantitative genetic, and population genetic approaches(2004) Symonds, Victor Vaughan; Lloyd, Alan M.Item Germination studies in Arabidopsis thaliana and Sinapis arvensis : genetical and ecological perspectives(2013-08) Morrison, Ginnie Denise; Linder, C. RandalThe environment can exert strong selective pressures on an organism. When selective pressures on traits differ between environments local adaptation may occur. If there is gene flow between the environments, local adaptation may be slowed or prevented. In plants, particularly weedy ephemerals, germination is a life-history trait that can be a strong determinant on fitness. In this dissertation, I explore the germination traits of two weedy Brassicaceae species, Arabidopsis thaliana and Sinapis arvensis, having populations in different habitats to determine whether germination traits within and between populations vary based on environmental conditions and to assess the extent of local adaptation. In Chapter 1, I assessed which genomic regions of A. thaliana were associated with differences in germination traits due to genotype-by-environment interactions. I performed a genome-wide association study using 100 natural accessions of A. thaliana under four light and nutrient combinations. I found 20 single nucleotide polymorphisms significantly associated with different environments, but none associated specifically with genotype-by-environment interactions. In Chapter 2, I assessed germination traits of S. arvensis collected from agricultural and non-agricultural habitats in the Bitterroot Valley of Montana. I discovered that the agricultural collection studied exhibited significantly different germination timing and amounts than the non-agricultural collections, which were statistically indistinguishable from each other. I also found evidence of a strong maternal effect on germination traits. In Chapter 3, I tested whether patterns of genetic variation between agricultural and non-agricultural collections of S. arvensis supported local adaptation to the two habitats even in the face of gene flow. While I expected to see some genetic differentiation between habitats, as seen in Chapter 2, no genetic differentiation was detected and markers putatively under selection were not associated with a particular habitat. I discuss why this might have occurred even though I have evidence for genetically-based phenotypic differentiation between agricultural and non-agricultural populations of S. arvensis.Item Histone H1 in Arabidopsis thaliana(2014-05) Jones, Ashley Loray; Sung, SibumHistone H1, or linker histone, are unique histones that bind to the nucleosome to facilitate higher order chromatin structure. The linker histones, when compared to the core histones that make up the nucleosome, are poorly understood especially in plants. Linker histones are vital for plant development as well as for cell cycle regulation, sharing many qualities with animal linker histones. In this report, the first two parts introduce the current literature of H1, including result from non-plant systems, and the third section is a research proposal describing a research project to elucidate the roles of linker histones on the regulation of FLOWERING LOCUS C (FLC) in Arabidopsis thaliana.Item In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana(2012-07-16) Johnson, CarolChloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin and to have been transferred from a symbiont's genome to host genome via lateral transfer. Subsequent duplication of the initial FtsZ gene gave rise to the FtsZ1 and FtsZ2 genes and protein families in eukaryotes. These proteins co-localize mid-chloroplast to form the Z-ring. Z-ring assembly initiates chloroplast division, and it serves as a scaffold for other chloroplast division proteins. Little is known, however, about the FtsZ protein subunit turnover within the Z-ring, the effects of accessory proteins on Z-ring turnover assemblies, as well as the in vivo ultrastructure of the Z-ring in plants. To investigate the Arabidopsis thaliana FtsZ subunit turnover rate within the Z-ring, a section of the Z-ring in the chloroplasts of living plants expressing fluorescently tagged FtsZ1 or FtsZ2 proteins was photobleached and the recovery rate was monitored. The results show that the fluorescence recovery half times for the FtsZ1 and FtsZ2 proteins are 117s and 325s, respectively. This is significant as these data mirror their differences in GTP hydrolysis rates. To elucidate in vivo structure and ultrastructure of the Z-ring, a protocol was established that maintained fluorescence during high pressure freezing, freeze substitution and low temperature embedding. Afterwards, a correlative microscopy approach was employed to visualize and identify fluorescently labeled puncta, circular structures, at the light microscopy level. These puncta were further resolved as mini-rings using optical microscopy eXperimental (OMX) superresolution microscopy. Electron microscopy (EM) analysis imaged mini-rings and filament assemblies comprised of dense subunits. Electron tomography (ET) showed mini-rings composed of protofilaments.Item Investigations into extracellular nucleotide-based signaling mechanisms in plants(2004-08) Jeter, Collene Renee, 1968-; Roux, Stanley J.Item Investigations into the role of cGMP in mediating the effects of extracellular nucleotides on root hair growth in Arabidopsis thaliana(2009-08) Steere, Barbara A.; Roux, Stanley J.; Clark, Gregory B.The eATP pathway begins a cascade of events which includes the involvement of nitric oxide synthase (NOS) and nitrate reductase (NR) in the production of nitric oxide (NO). Research has shown that SNAP (S-nitroso-N-acetylpenicillamine) and NONOates (diazeniumdiolates) promote the availability of NO and, with the addition of guanylate cyclase, form cyclic guanine monophosphate (cGMP), and root hair growth is promoted. Phosphodiesterases (PDE) break down the cGMP and agents such as IBMX and Viagra inhibit the PDEs thereby inhibiting root hair growth. Several questions remain to be answered. How much cGMP is necessary for the promotion of root hair growth? Is there an optimal concentration of cGMP which stimulates root hair growth, above which is inhibitory, or below which is ineffective? Is there a “non-hydrolyzable analog” of cGMP which is more effective at promoting root hair growth? Is it possible to see inhibition of root hair growth with exposure to a known inhibitor, such as ATPγS, and then reverse the inhibition with a “non-hydrolyzable analog” of cGMP? Answering these vi questions is the substance of this research and the answers will provide direction and understanding to the growth-promoting and regulatory role eATP plays in signal transduction pathways in plants. With the hypothesis asking whether the effects of NO on root hair growth is cGMP-dependent or cGMP-independent we found that there is no consistent concentration of non-hydrolyzable cGMP analog which promotes root hair growth. Additionally we found that the 8-Br-cGMP analog promotes root hair growth more consistently in Arabidopsis thaliana than its counterpart, dibutyryl cGMP. We substantiated previously published results showing an inhibition of root hair growth when root hairs were exposed to high concentrations of ATPγS. Based on these results we believe the promotion of root hair growth in Arabidopsis thaliana to be mediated independently of cGMP.Item The MAR1 transporter of Arabidopsis thaliana has roles in aminoglycoside antibiotic transport and iron homeostasis(2009-08) Conte, Sarah Schorr; Lloyd, Alan M.Widespread antibiotic resistance is a major public health concern, and plants represent an emerging antibiotic exposure route. Recent studies indicate that crop plants fertilized with antibiotic-laden animal manure accumulate antibiotics, however, the molecular mechanisms of antibiotic entry and subcellular partitioning within plant cells remain unknown. Here we report that mutations in the Arabidopsis locus Multiple Antibiotic Resistance (MAR1) confer resistance, while MAR1 overexpression causes hypersensitivity to multiple aminoglycoside antibiotics. Resistance is highly specific for aminoglycosides and does not extend to antibiotics of other classes, including the aminocyclitol, spectinomycin. Yeast expressing MAR1 are hypersensitive to the aminoglycoside, G418, but not to chloramphenicol or cycloheximide. MAR1 encodes a protein with 11 putative transmembrane domains with low similarity to ferroportin1 from Danio rerio. A MAR1:YFP fusion protein localizes to the chloroplast, and chloroplasts from plants overexpressing MAR1 accumulate more of the aminoglycoside, gentamicin, while mar1-1 mutant chloroplasts accumulate less than wild type. MAR1 overexpression lines are slightly chlorotic, and this chlorosis is rescued by application of exogenous iron. MAR1 expression is also downregulated by low iron. Taken together, these data suggest that MAR1 is a plastid transporter that is likely to be involved in cellular iron homeostasis, and allows opportunistic entry of multiple antibiotics into the chloroplast. mar1 mutants represent an interesting example of plant antibiotic resistance that is based on the restriction of antibiotic entry into a subcellular compartment. Knowledge about this process – and other processes of antibiotic entry – could enable the production of crop plants that are incapable of antibiotic accumulation, aid in development of phytoremediation strategies for decontamination of water and soils polluted with antibiotics, and further the development of new plant-based molecular markers. The work described here also contributes to our understanding of how plants interact with the antibiotics they encounter, both in the laboratory (where aminoglycosides such as kanamycin are used heavily to select for transgenics) and in the natural environment.Item Molecular Mechanisms of Arabidopsis Resistance to Green Peach Aphid, Myzus Persicae(2014-11-26) Lei, Jia XinAphid, a specialized phloem sap-feeding insect, is one of the major pests of a number of economic important crops, including soybean, cotton, and maize. Aphid can have devastating effects on the crop production by limiting plant growth, as well as serving as vectors for disease. Therefore, research on identification of plant resistance mechanisms to aphid is important for crop improvement. Here, I have developed a system consisting of Arabidopsis thaliana and green peach aphids (Myzus persicae) to study plant-aphid interaction. In my research project, the role of BIK1 was investigated in Arabidopsis infested with the green peach aphid. Loss of BIK1 function adversely impacted aphid settling, feeding and reproduction. Relative to wild-type plants, bik1 displayed higher aphid-induced H2O2 accumulation and more severe lesions, resembling a hypersensitive response (HR) against pathogens. Basal as well as induced salicylic acid and ethylene accumulation were in in the bik1 mutant. Intriguingly, elevated salicylic acid levels did not contribute to the HR-like symptoms or to the heightened aphid resistance associated with the bik1 mutant. Elevated ethylene levels in bik1 accounted for an initial, short-term repellence. Introducing a loss-of-function mutation in the aphid resistance and senescence-promoting gene PHYTOALEXIN DEFICIENT4 (PAD4) into the bik1 background blocked both aphid resistance and HR-like symptoms, indicating bik1-mediated resistance to aphids is PAD4-dependent. Taken together, Arabidopsis BIK1 confers susceptibility to aphid infestation through its suppression of PAD4 expression. Furthermore, the results underscore the role of ROS and cell death in plant defense against phloem sap-feeding insects.Item Role of casein kinase 2 (CK2) alpha subunits in the growth and development of Arabidopsis thaliana(2015-05) Mulekar, Jidnyasa; Huq, Enamul; Browning, Karen; Lloyd, Alan; Roux, Stanley; Sung, SibumCasein kinase 2 (CK2) is a conserved Ser/Thr kinase present in all organisms. It is a tetrameric protein composed of two alpha and two beta subunits. In Arabidopsis, there are 4 genes encoding for each alpha (catalytic) and beta (regulatory) subunit of CK2. The role of CK2 alpha subunits in the growth and development of Arabidopsis was studied through characterization of T-DNA insertion mutants (for α1, α2, α3) and RNAi approach (for α4). Results show that all 4 CK2 alpha subunits positively regulate flowering under both short day and long day conditions, possibly in a functionally redundant manner. Elevated levels of FLC and reduced levels of SOC1 in the CK2 alpha triple mutant compared to wild type (Col-0) suggest that the alpha subunits possibly act through the autonomous pathway to regulate flowering. The alpha subunits also synergistically promote inhibition of seed germination and cotyledon greening in the presence of ABA and salt (NaCl). I also found that the CK2 alpha subunits redundantly control lateral root formation in Arabidopsis. CK2 has been shown to be involved in light signaling and phosphorylates the regulatory proteins HY5, HFR1 and PIF1 in the pathway. PIF1 belongs to a family of bHLH transcription factors (Phytochrome Interacting Factors) which function as repressors of photomorphogenesis. In the presence of light, activated phytochromes (red/far red light photoreceptors) translocate to nucleus and interact with PIFs, which leads to rapid phosphorylation of PIFs followed by their degradation through the 26S proteasome. An in vitro kinase assay was performed to test whether CK2 phosphorylates all the PIF family proteins. Results show that CK2 phosphorylates PIF1, PIF3 and PIF5 but does not phosphorylate PIF4, PIF6 and PIF7. Up to 10 putative CK2 sites in PIF3 protein were mapped and mutated however the mutant protein was still significantly phosphorylated by CK2. The CK2 phospho-deficient mutant of PIF1 (PIF1-7M) showed reduced homo-dimerization capacity than the wild type in yeast two hybrid assays. However, further studies are required to access the functional significance of it. Overall, this research uncovers novel functions for CK2 alpha subunits in the growth and development of Arabidopsis thaliana.Item Studies on the chromatographic behavior and distribution of sterols from Arabidopis thaliana(Texas Tech University, 1998-08) Lopez, MonicaStudies were performed on the chromatographic behavior of sterols in thin-layer chromatography, gas-liquid chromatography, high performance liquid chromatography, argentation chromatography and flash chromatography. The structure of the sterol was found to be influenced by its mobility in the various chromatographic systems tested. For instance, C-4 substitution dramatically affected sterol mobility in TLC whereas, C-24 substitution dramatically affected sterol mobility in GLC and HPLC. Anatomical parts were separated from the mature plant of Arabidopsis thaliana for sterol analysis The mixture of phytosterols was found to differ in the five plant parts. The sterol composition of A. thaliana was found to operate a typical tracheophytic cydoartenol-sitosterol based pathway. Except for the fruits, carbon fluxed from cycloartenol to predominantly 24-ethyl sterols. In the fruits, a novel 24-desalkyl sterol was found to accumulate in the tissues. Based on GLCMS characterization, the sterol was tentatively identified as cholesta-9(11), 24-dienol.Item Studies on the purification and characterization of the (s)-adensyl-l-methionine sterol methyl transferase from Arabidopsis thaliana(Texas Tech University, 1999-08) Zhou, WenxuThe sterol methyl transferase gene from Arabidopsis thalicma that encodes for the 24 consecutive C-methylation of the A -bond of the sterol side chain was introduced into plasmid pET15b and the resuhing native protein was overexpressed in BL2i(DE3) host cells under control of a T? promoter. The enzyme was partially purified by anion exchange and gel filtration chromatography. GPC chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a native molecular weight of 158.000 Dahons and was tetrameric. Studies on product formation using sterols native to A. thaliana, cycloartenol (K^^^^^^ = 46 ^M and y^^^^ppj = ^ pmo/min/mg) and 24(28)-methylene lophenol 0^m{app) " ^^ I^^ ^"^^maxíapp) ^ ^-^^ pmol/min/mg), as substrates incubated with a soluble SMT preparation confirmed that the first and second C-methyl transfer reactions proceeded stereoselctively to a single product 24(28)-methyIene cycloartanol and 24(28)Z-thyIidene lophenol, respectively. From a study involving incubation of a series of related substrates that were structurally similar to cycloartenol and 24(28)methylene lophenol with a soluble enzyme preparation of the SMT, the sterol features recognized by the SMT for binding were established: a free 3P-hydroxyl group, a side chain with a A" -bond and a planar nucleus. An unexpected fmding was that zymosterol (K^(Û„„; ^28 |iM and yjnnxfappj ^11^ pmol/min/mg), the preferred sterol substrate for the yeast SMT was more effective in binding to the plant SMT from A. thaliana than cycloartenol. From a kinetic study involving a series of high energy intermediate and product analogs, we observed that 24p-methyl sterols inhibited Cmethylation consistent whh the proposed mechanism of a iS/-face attack ("Steric-EIectric Plug model" and that the first and second C-methyl transfer reactions utilize different substrates, but nonetheless undergo transformation from the same binding site. Sitosterol, but not cholesterol was bound productively to the SMT and inhibited enzyme activity with competitive-type kinetics relative to ehher cycloartenol or 24(28)-methyIene lophenol as substrates. Using a ftision construct containing a His Tag and a native construct, a set of shedirected mutants have been generated at Trp89 and Tyr85, aromatic amino acid residues assumed to be contained whhin the sterol binding site of this enzyme. Whereas no change in ehher the product distribution or sterol specificity of these mutants was observed, a change in catalytic efficiences was established in several cases. From the combination of resuhs, the interrelationships of substrate fiinctional groups whhin the active center could be approximated showing that a single SMT can give rise to mono or doubly alkylated side chains by the same reaction mechanism.Item Telomerase activator1: a zinc-finger protein that acts synergistically with auxin to control telomerase expression in Arabidopsis thaliana(Texas A&M University, 2006-04-12) Ren, ShuxinTelomerase 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.Item Telomerase Regulation in Arabidopsis thaliana(2012-10-19) Nelson, AndrewTelomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct.Item The dna ?saw puzzle??ructure model: the case studies of the rice and yeast genomes(2009-05-15) Liu, Yun-HuaHow does DNA make the abundant and diverged life world? To address this question, a DNA ?Jigsaw Puzzle? structure model was proposed and first tested by comprehensively analyzing the genome of the model dicot plant, Arabidopsis thaliana. However, it is unknown whether this model is held in other species. Here we report the studies of the DNA structure model using the monocot plant model species, rice (Oryza sativa), and the single-celled model species, yeast (Saccharomyces cerevisiae). Analyses of the genomes sequenced so far revealed that the genome of an organism consists of a limited number of sequence-specialized, so-called fundamental function elements. For a higher organism, these elements often include genes (GEN), retro-transposable elements (RTE), DNA transposable elements (DTE), simple sequence repeats (SSR) and low complex repeats (LCR). Datasets were developed for RTE, DTE, SSR, LCR and GEN as well as genes categorized into different function categories from the sequences of the rice and yeast genomes using appropriate window sizes. The datasets were subjected to statistical analyses to test the DNA ?Jigsaw Puzzle? structure model in terms of the unambiguousness, correlation, uniqueness and selection of their genome-constituting element arrays. The analyses were conducted with a series of window sizes of the sequences at both the whole genome and individual chromosome levels, both including and excluding the centromeric regions. The results showed that all fundamental function elements of the genomes as well as the genes categorized into different function categories were arrayed in the genomes in an unambiguous manner resembling linear ?Jigsaw Puzzles? at the whole genome and/or individual chromosome levels, no matter whether the centromeric regions were included or excluded. The analyses revealed that arraying of the genomic elements was correlated significantly and uniquely for each chromosome and each species. This further confirmed the non-random arraying characteristic of the genomic elements for the DNA ?Jigsaw Puzzle? structure model and suggested that the DNA ?Jigsaw Puzzle? structure is unique for an organism, which has probably resulted from natural selection. These results unambiguously support the hypothesis of the DNA ?Jigsaw Puzzle? structure model. Since the content, arraying and interaction pattern of the fundamental function elements were shown to be unique for each organism, variations of an organism in its DNA ?Jigsaw Puzzle? array would lead to phenotypic variations, thus resulting in different organisms. Moreover, the fundamental function elements constituting a genome, as the four nucleotides (A, T, G and C) of DNA, could be arrayed into an infinite number of DNA molecules, thus giving different forms of organisms. Therefore, the DNA ?Jigsaw Puzzle? structure model would provide a novel, but convincing explanation for the abundance, diversity and complexity of living organisms in the world.