Doctor of Philosophy, The Ohio State University, 2006, Molecular, Cellular, and Developmental Biology

The circadian oscillator of Arabidopsis thaliana builds upon interlocked transcriptional feedback loops onto which a diverse array of period-affecting factors are incorporated to culminate into a 24-hour periodicity. A subset of the period-affecting factors function in light input pathways which are specifically connected to the clock, thereby modulating circadian period in a light-dependent manner. A novel F-box protein, ZEITLUPE (ZTL) which controls circadian period in a fluence-rate dependent way, falls into this category. The primary focus of this dissertation is to characterize the light-regulated proteolytic pathway mediated by ZTL. At first we identified the SCFZTL complex in planta and established that the formation of SCFZTL complex is important for proper regulation of circadian clock. SCFZTL complex targets an essential oscillator component, TOC1 for its proteasome-dependent degradation. TOC1/PRR1 is the founding member of a small gene family of Pseudoresponse Regulators (PRRs) which consists of additional four genes (PRR3,PRR5,PRR7 and PRR9). The transcriptional regulations among PRR members partially define the interlocked transcriptional feedback loops which are at the core of central oscillator. In this study we characterized the expression pattern of all PRR members and identified two post-translational regulatory events which are shared by four PRR members (PRR1/3/5/7): phosphorylation and proteasome-dependent protein turn over. In addition, we identified PRR5 as a novel interactor of ZTL. In order to further characterize biochemical properties of ZTL and more broadly, to better understand the ZTL-mediated light signal transduction pathway which is specifically coupled to circadian clock, we took both forward and reverse genetic approaches. First, we dissected the roles of three distinctive domains of ZTL in the regulation of circadian period and photomorphogenesis. We found that the C-terminal F/KELCH domain could act together to alter circadian pe (open full item for complete abstract)

Committee: David Somers (Advisor); Greg Armstrong (Other); David Bisaro (Other); David Mackey (Other) Subjects: Biology, Plant Physiology

Doctor of Philosophy, The Ohio State University, 2005, Horticulture and Crop Science

The objective of this study was to increase the resistance of crops (tobacco and tomato) to water deficit stress. To achieve this goal transgenic plants were generated using two genes, a putative tomato type I inositol 5 polyphosphatase (5PTase) that terminates inositol 1,4,5 trisphosphate (IP3) signaling and ABRE binding factor ABF4 derived from Arabidopsis. Inositol 1,4,5 triphosphate (IP3), as one of phosphoinositides, is known to transduce a stress signal by changing its level in response to water deficit, osmotic stress, and low temperature in plants. Recently it was shown that type I inositol 5 phosphatases (5PTases), At5PTase1 and AtIP5PII/At5PTase2, regulate IP3 level in plant like in animals and that up-regulation of these genes decreases IP3 levels which leads to a reduction in the expression of ABA-/drought-responsive genes in Arabidopsis. On the basis of sequence similarity to Arabidopsis 5PTases, four tomato cDNAs (Le5PT1-4) that encode putative tomato type I 5PTase proteins were identified. Predicted protein sequences of identified Le5PTs had conserved catalytic domains that are required for 5PTase enzyme activity. Two clones, Le5PT1 and Le5PT2 were similar to AtIP5PII/At5PTase2 and At5PTase1, respectively. The expression of Le5PT1 was down-regulated in early time point under dehydration, NaCl, and exogenous ABA treatment, indicating that Le5PT1 may play a negative role in stress signaling. Transgenic tobacco plants with 35S:Le5PT1 did exhibit weak expression of the drought inducible gene, NtERD10B, but did not show correlation with resistance to water deficit stress. AtABF4, a bzip transcription factor, is known to induce the expression of ABA-responsive genes. The expression of Arabidopsis ABF4/AREB2 gene under the control of guard cell specific KST1 promoter was shown to significantly increase drought tolerance in tobacco and tomato plants. The transgenic plants exhibit significantly lower water loss per unit leaf area compared to wild type plants. (open full item for complete abstract)

Committee: James Metzger (Advisor) Subjects: Biology, Plant Physiology

Doctor of Philosophy, The Ohio State University, 2002, Horticulture and Crop Science

Environmental factors such as light and temperature play an important role in plant development. The bolting response associated with flowering in Arabidopsis and other rosette species is regulated by gibberellin (GA). Presumably there are one or more blocks in the GA biosynthetic pathway that are removed following induction of flowering by either long-days (LD) or vernalization. The objective of this research was to identify steps in biosynthesis regulated by photoperiod and vernalization in Arabidopsis thaliana by comparing the biological activities of native GAs and GA precursors under inductive and non-inductive condition. A T-DNA tagged dominant mutant with a cold requirement line (CR) and a recombinant inbred line selected for a LD requirement (CS933) were used. Quantitative analysis of the content of kaurenoic acid (KA) in these two environmental responsive types when plants were subjected to inductive conditions did not support the hypothesis that the GA biosynthesis pathway is regulated in the conversion of KA to 7-OH KA in Arabidopsis. For 20 carbon GAs, a common feature that distinguished between active and inactive compounds was the oxidation state of C-20. GA precursors and GAs containing a C-20 methyl group (GA12) were not active, while GAs containing a C-20 aldehyde (GA24 and GA36) had substantial biological activity when applied to non-induced plants of both response types. This result suggests that the limiting step in GA biosynthesis pathway is located at the step in which the C-20 carbon is oxidized. In addition, for both 19 and 20 carbon GAs, C13 hydroxylation resulted in reduced biological activity when compared to the non-C13 hydroxylated homologs. These results suggest that 1) under non-inductive conditions C-20 oxidation is the rate-limiting step in the synthesis of bioactive GAs; 2) both vernalization and LD regulate GA biosynthesis at the same control point; 3) the non-C13 hydroxylation pathway leading to GA4 may be more important in regula (open full item for complete abstract)

Committee: James Metzger (Advisor) Subjects: Biology, Plant Physiology

Master of Science (MS), Ohio University, 2007, Plant Biology (Arts and Sciences)

Xyloglucans are the major hemicellulosic polysaccharides in the primary cell walls of dicots and non-graminaceous monocots but small amounts are also seen in the walls of grasses. They serve as cross-linkers of cellulose microfibrils and maintain the structural integrity of the cell.Xyloglucan biosynthesis mechanism is well understood and many biosynthetic genes have been identified in dicots. However this process is not known in grasses where structural differences in xyloglucan exist. Thus, this study aimed to investigate xyloglucan biosynthesis in grasses by functionally characterizing some xyloglucan biosynthetic genes, particularly the xylosyltransferases from wheat and rice. Wheat (TaGT34-7) and rice (OsGT34-3) homologs of Arabidopsis xyloglucan-xylosyltransferase (AtXT1) gene were previously identified using a bioinformatic script developed by Faik et al. (2006). The full-length cDNAs encoding TaGT34-7 and OsGT34-3 proteins were expressed in Pichia pastoris and Drosophila S2 expression systems, but the proteins did not show xylosyltransferase activity in Pichia pastoris and a low activity in Drosophila S2 cells. Further optimization is required to fully identify the function of these two proteins. In another study, the endosperm developmental phases were identified in developing wheat seeds using sectioning and light microscopy. Cell walls of wheat endosperm have ~70% (w/w) of arabinoxylan (AX), 20% (w/w) of mixed linked glucan (MLG), and only 5% (w/w) of cellulose. MLG and AX are deposited during cellularization and differentiation phases of the endosperm development, respectively. To identify the putative glycosyltransferase genes involved in the biosynthesis of these polymers (AX, MLG), it was necessary to first identify the endosperm developmental phases namely cellularization and differentiation phases at which they are deposited. Fresh wheat seeds were collected and their endosperm developmental phases were investigated using sectioning and light micros (open full item for complete abstract)

Committee: Ahmed Faik (Advisor) Subjects: Biology, Plant Physiology

Master of Science (MS), Ohio University, 2005, Plant Biology (Arts and Sciences)

The restoration of the American chestnut to U.S. forests is currently a subject of much interest. Questions remain about where on the landscape chestnut should be replanted, and the ecophysiological capacity of chestnut at different ontogenetic stages. The goals of the present study were to assess the physiological and leaf characteristics of chestnut and to quantify these characteristics at the seedling, sapling, and mature growth stage. The photosynthetic performance, leaf mass per area, and leaf nitrogen content of chestnut were assessed at sites in Wisconsin and Ohio. Seedlings maximized photosynthesis under high light conditions, a practical result for foresters replanting chestnut. The physiology and leaf characteristics of seedlings and saplings in the understory were similar. Photosynthesis and leaf nitrogen were maximized at the top of the canopy, declining with stand height and light availability. These results will be useful in modeling the carbon dynamics of mature American chestnut forests.

Committee: Kim Brown (Advisor) Subjects: Biology, Plant Physiology

Master of Science (MS), Ohio University, 2003, Plant Biology (Arts and Sciences)

The enzyme invertase contributes to sugar unloading, pathogen defense,differentiation and development in plants. One soluble invertase cDNA was cloned using RACE in this study. Its sequence structure and its sequence similarity to that of other invertases suggest that the full-length 2237bp cDNA (PsI-1 cDNA), represents a functional vacuolar invertase. It is strongly expressed in sink organs such as young stem,root tip, and flower buds. Its expression is up-regulated by GA. Two genomic clones(clone 8 and clone 4) were identified. Clone 8 is believed to contain the PsI-1 gene based on sequence similarity. The presence of an array of potential cis-acting elements in the 5'upstream sequence of PsI-1 suggest that its expression can be adjusted to suit a variety of developmental and environmental needs. Comparison of sequence similarities and gene structure suggests that the invertase-homologous sequence in clone 4 may have originated from an invertase gene/pseudogene different from PsI-1.

Committee: John Mitchell (Advisor) Subjects: Biology, Plant Physiology

Master of Science, Miami University, 2005, Botany

This study was conducted to examine the physiological effect of agriculturally relevant levels of cadmium on field and greenhouse grown soybean (Glycine max). Cadmium levels of 0, 2.5, 5, and 10 ig Cd/g dry wgt soil were selected. Measurements of instantaneous gas exchange, carbon assimilation response to leaf internal CO2 concentration, sap flow, chlorophyll fluorescence, growth parameters and cadmium levels in leaf and soil were conducted throughout the soybeans life span. There was a significant increase in total sap flow between the control and 10 ig Cd/g soil plants. Peak sap flow was found to significantly increase in the 10 ig Cd/g treatment. Time of initial sap flow was also significantly delayed in Cd treated plants. Additionally, instantaneous transpiration was significantly increased during one sampling period in the 10 ig Cd/g treatment. The changes in sap flow rates following low-level Cd treatment may be attributed to damaged and non-responsive stomata.

Committee: Alfredo Huerta (Advisor) Subjects: Biology, Plant Physiology

Doctor of Philosophy, Miami University, 2003, Botany

The four objectives of this study were to: 1) Determine the usefulness of the plastochron index (PI) in S-deficiency studies in bean (Phaseolus vulgaris var. Bush); 2) Characterize the early onset of sulfur (S) deficiency; 3) Determine if higher light exacerbates S deficiency symptoms; and 4) Determine if glutathione (GSH) limitation is associated with onset of S deficiency symptoms. The PI can decrease variability in data, yet few physiological studies have employed it because stress often disrupts underlying PI assumptions of: early exponential growth, similar leaf relative growth rates (RGR), and similar plastochrons. In this study, while S-stressed plants showed exponential growth, RGR declined and plastochron increased. Calculation of the RGR-induced error suggested that the deviations had little negative impact on results. To address unequal plastrochrons, the curvilinear PI-time relationship was used to calculate Plastochron Index Derived Days (PID), i.e., chronological age predicted from PI. Using PID greatly decreased variability of physiological data (e.g., increased r2 for CO2 assimilation (A) vs. time from 0.2 to 0.7) and minimized subjective plant selection. Sulfur limitation caused stunted morphological development by 15 days after planting (DAP); decreased total leaf S and A by 16 DAP; and decreased %GSH in reduced form, maximal A (A max), Rubsico carboxylation efficiency (CE), Fv/Fm, dry weight, and chlorophyll concentration, but increased the CO2 compensation point (Γ), by 21 DAP. Higher light intensity (200 μmol m-2s-1) sped the onset and amplified the magnitude of S deficiency symptoms in bean. Higher light slowed morphological development, decreased A max, CE, %GSH, chlorophyll, dry weight, Fv/Fm, and increased Γ, but 60 μmol m-2s-1 effected these symptoms less or not at all. This project hypothesized a linkage between GSH-dependent photoprotection and onset of S deficiency symptoms. Surprisingly, neither supplied S nor light level had any effect (open full item for complete abstract)

Committee: Alfredo Huerta (Advisor) Subjects: Biology, Plant Physiology

Doctor of Philosophy, Case Western Reserve University, 1996, Biology

To identify genes involved in auxin conjugate hydrolysis, we selected Arabidopsis thaliana mutants resistant to growth inhibition by IAA-phenylalanine (IAA-phe). Two IAA conjugate resistant mutants were isolated: icr1 and icr2. Neither of these strains appears to be a conjugate uptake mutant. Dose-response experiments have shown that the mutants are approximately 2- to 3-fold more resistant to IAA-phe and 3-fold more resistant to IAA-alanine than wild-type plants. Although IAA-aspartate is not a strong growth inhibitor of Arabidopsis, the mutants are ∼2-fold more resistant than wild-type plants at 250-1000 mM of IAA-aspartate. Conjugate hydrolysis experiments indicate that icr1 and icr2 hydrolyte at least as well as wild-types. The icr1 mutation is a homozygous, incompletely dominant trait and maps to the top of Chromosome V. The Arabidopsis strain carrying icr1 demonstrates two additional mutations: delayed germination and early flowering. Analysis of a backcross of icr1 to Ws showed that additional traits are not caused by icr2. The late germination trait demonstrates some genetic linkage to the icr1 locus. We have also used a microbial selection scheme as a separate approach to isolating the genes for conjugate hydrolysis. From an Arabidopsis cDNA library we selected a transformed yeast strain able to utilize IAA-phe as a source of phenylalanine. The 1800 bp cDNA from this transformed strain was found to be an exact match to Arabidopsis actin2. Plasmid loss was shown to be accompanied by an inability of the yeast to utilize IAA-phe. Furthermore, down-regulation of the yeast promoter driving actin expression resulted in an inability of the transformed yeast to grow on IAA-phe, indicating expression of the actin is required for growth on this conjugate. IAA-aspartate and IAA-valine also supported growth of the appropriate yeast strains transformed with Arabidopsis actin. Overexpression of yeast actin also allowed auxotrophs to survive on conjugate, indicating that (open full item for complete abstract)

Committee: Christopher Town (Advisor) Subjects: Biology, Plant Physiology

Doctor of Philosophy, Case Western Reserve University, 1993, Biology

Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by alpha-methyltryptophan (αMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild-type, both as plants and callus. However mutant plant growth is unaffected by 25 μM αMT and mutant callus growth by 50 μM αMT, concentrations which completely inhibit the growth of wild-type plants and callus respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 μg/g freshweight respectively and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 μg/g fresh weight, respectively. Anthranilate synthase activity levels (nmol/mg protein hr) in crude extracts from whole plants are 3.09 ± 0.54 in amt-1 and 1.32 ± 0.21 in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 in amt-1 and 7.74 ± 1.58 in wild-type. Enzyme extracts from plants and callus are inhibited by L-tryptophan; IC50 values are 4.6 ± 0.6 μM for amt-1 plants and 1.5 ± 0.5 μM for wild-type plant extracts and 6.9 ± 0.4 and 3.4 ± 0.5 for mutant and wil d-type callus extracts respectively. The mutation segregates as a single nuclear allele, shows incomplete dominance, and maps to the top of Chromosome V within 1.5 map units of ASA1, a gene for the large subunit of anthranilate synthase. The concommitant increases in both anthranilate synthase activity and its IC50 for tryptophan suggest that the mutation amt-1 resides either in one of the anthranilate synthase structural genes or in a gene whose product modifies anthranilate synthase activity

Committee: Christopher Town (Advisor) Subjects: Biology, Plant Physiology