Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3354. Adviser: Thomas W. Jacobs. Includes bibliographical references (leaves 143-158) Available on microfilm from Pro Quest Information and Learning.

Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

The use of neem products to control the European corn borer, Ostrinia nubilalis (Hubner), in sweet corn was investigated. Three years of field studies were conducted comparing various foliar-spray applications of a neem seed kernel extract (NSKE), azadirachtin (AZA, the active ingredient of neem extracts), and Ambush (a synthetic pyrethroid). The results included: (1) NSKE sprayed prior to artificial infestation of the corn plants provided excellent protection (comparable to Ambush) against borer damage and greatly reduced larval populations. (2) Neem extract formulations from Safer Ltd. were the most effective. (3) The rate of application or number of applications were not determining factors in altering the efficacy of neem products. (4) Exceptional weather conditions (high temperatures, dry conditions) may be detrimental to the efficacy of foliar applied neem products. (5) Some neem treatments were found to increase corn stalk breakage possibly by altering the behavior of the larvae. Laboratory evaluations using no-choice leaf-disk bioassays showed the antifeedant action of AZA and the negligible effect of PBO (piperonyl butoxide) and Citowett (used in field formulations) on the feeding behavior of 3rd instar larvae. Growth and development studies of O. nubilalis fed a diet containing 0, 1, 3, 10, 30, 100, and 1000 $\mu$g NSKE g$\sp{-1}$ diet were conducted. 10 $\mu$g NSKE g$\sp{-1}$ diet (50 ng AZA g$\sp{-1}$ diet) fed continuously to the larvae were larvicidal and concentrations as low as 3 $\mu$g NSKE g$\sp{-1}$ diet (16.6 ng AZA g$\sp{-1}$ diet) significantly reduced larval, pupal and adult weights, increased the larval and pupal periods, decreased adult emergence and caused wing deformities. AZA content of the extracts was found to be a critical factor in the IGR (insect growth regulator) effects of NSKE. (Abstract shortened by UMI.)

Fenitrothion (0,0-dimethyl-0-(4-nitro-m-tolyl) phosphorothioate) was absorbed by germinating seeds of eastern white pine; Pinus strobus L. (Moench) Voss, white spruce; Picea-qlauca L., and yellow birch; Betula alleghaniensis L., from an aqueous solution containing 10 ppm of the pesticide. Metabolism of the pesticide was examined in young seedlings with OC14H3 labelled fenitrothion. Fenitrothion was shown to be detoxified by dealkylation in white pine via a glutathione dependent S-alkyl transferase enzyme to form desmethyl fenitrothion and S-methyl glutathione. Dealkylation was also found in white spruce and yellow birch seedlings. The pesticide was activated to form fenitro-oxon, which is more toxic than the parent compound. The desmethyl form was shown to be reactivated via alkylation by fenitrothion to form S-methyl fenitrothion which is a more potent cholinesterase inhibitor than the parent compound. Treatment of the seeds at higher concentrations of 1000 ppm fenitrothion inhibited germination of yellow birch seeds but not white pine or spruce. Severe depletion of tissue glutathione levels was shown to be related to toxicity in yellow birch which absorbed much higher levels of pesticide than the two coniferous species. Fenitrothion and seven derivatives of the compound were tested for mutagenicity via alkylation of replicating DNA in an indicator strain of Salmonella typhimurium, sensitive to base-pair substitutions caused by alkylating agents. No mutagenic activity was evident for fenitrothion or its derivatives in this system.

This study postulates that the symbiosis between red clover, Trifolium pratense L., and the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenck & Smith, enhances secondary phenolic metabolism and nitrogen assimilation. To test this hypothesis, the four objectives set forth were to determine the impact of AM colonization and/or rhizobial inoculation in red clover on the: (1) growth parameters; (2) nitrogen assimilation, via the activity of glutamine synthetase (GS, E.C. 6.3.1.2), total N content and soluble protein concentration; (3) contents of isoflavonoids (e.g. genistein, daidzein, biochanin A, and formononetin); and (4) contents of these four isoflavonoids over time and with a hormonal factor, jasmonic acid (JA). To accomplish these objectives, four greenhouse experiments were performed. The overall results indicate that the colonized red clover plants, with 22% to 37% of AM root colonization, had enhanced shoot height as well as increased shoot and root dry masses. Co-inoculation with Rhizobium enhanced the growth parameters more than inoculation with either microsymbiont alone. The concentrations of micronutrients significantly increased in red clover with AM colonization and/or rhizobial inoculation, while the macronutrients did not vary to any great degree. (Abstract shortened by UMI.)

This thesis examines the accumulation of polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in vegetation in order to develop an interpretative scheme to determine deposition velocities of semi-volatile organic compounds (SVOCs) onto vegetation. PBDEs are flame retardants used in a variety of consumables. Although relatively non-volatile, these compounds have been quantified around the world extending to otherwise pristine ecosystems providing empirical evidence for their long-range transport. However, modeling studies indicate that their long-range atmospheric transport (LRAT) potential is at best moderate. However, recent modeling studies have suggested that vegetation may play an important role in their global distribution. PAHs are also ubiquitous contaminants. These can be released through both natural and anthropogenic sources. Some are considered highly carcinogenic and their potential impact on human health may be due to their association to particulates. Although their environmental fate is perhaps better understood, the processes involved in surface-air exchange, particularly with vegetation, have not been well documented. Spruce needles and atmospheric (gaseous and particulate-bound) PBDE and PAH concentrations were monitored bi-weekly from February 2004 to June 2005 to examine potential weather-related and seasonal effects. An efficient extraction method for PBDEs from spruce needles was developed. Finally, using measured concentrations, surface-air exchange was considered and a modeling concept was developed to determine deposition velocities to vegetation. Following their emergence, spruce needle PBDE and PAH concentrations increase gradually over time although decreasing briefly following snowmelt with a minimum coinciding with the following year's bud burst. Atmospheric concentrations of PBDE and PAH, both gaseous and particulate-bound, were linked to daily weather events. PBDE gaseous concentrations increased with temperature, whereas PAH concentrations were generally highest in the winter, likely reflecting increased emission. Analysis of air mass back trajectories and local wind directions revealed that particulate-bound PBDEs, along with both gaseous and particulate-bound PAHs originated from local sources, whereas gaseous PBDEs were likely from distant sources. Using measured atmospheric PBDE and PAH concentrations, particulate-gas partitioning was examined. Particulate-gas distributions correlated significantly with log KOA values and a significant temperature dependence was observed for most compounds considered, except the higher PBDE congeners. From compounds exclusively associated to particulates, the particulate-bound deposition velocities were calculated at 3.8 and 10.8 m/h for PBDEs and PAHs, respectively. The different vP values obtained for PBDEs and PAHs may indicate association with different particulates. Net gaseous transfer velocities correlated significantly with log KOA values and ranged from 2.4 to 62.2 m/h for PBDEs and from negligible to 75.6 m/h for PAHs. These derived values were then used to monitor PBDE and PAH accumulation in vegetation through time, and these agreed well with measured values. This study provides the necessary background for modeling PBDE and PAH transport between air and coniferous vegetation globally.

This doctoral thesis investigated the impact of the arbuscular mycorrhizal (AM) symbiosis, an ubiquitous and beneficial association between plant roots and soil fungi, toward plant growth, stress tolerance, and metal uptake in relation to extrinsic metal conditions ranging from low (e.g. trace) to high (e.g. toxic) exposure levels. The investigative strategy is divided into two main parts: (1) statistical meta-analysis and (2) experimental analysis. In the first part, an extensive literature review in the field of metal phytoremediation was conducted in order to construct a meta-dataset consisting of various plant physiological and soil ecological parameters which were ultimately extracted from nearly 30 published works. Meta-analytical statistical tools were then used to examine general trends and perspectives in metal phytoextraction and metal stress tolerance (Chapter 2), to establish an inherent role for the AM symbiosis therein (Chapter 3), and to discuss the potential for plant investment in symbiotic associations as an extrinsic stress tolerance strategy in complement to the plant's intrinsic stress resistance mechanisms (Chapter4). From these findings, a series of conceptual models were proposed depicting the plant growth and metal uptake in relation to increasing metal exposure levels by integrating the primary AM-induced mechanisms of 'enhanced uptake' and 'metal biosorption'. In the second part, in vitro root-organ (Chapter 5) and greenhouse culture systems (Chapters 6 and 7) were designed using the micronutrient zinc (Zn) as a typical metal contaminant to test various parameters of AM-plant growth and metal uptake, for which the proposed conceptual models were used as a framework for developing new hypotheses regarding plant-soil interactions. The methods and analytical techniques included Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) for the determination of soil- and plant-Zn concentrations, differential root staining and microscopic analysis for the assessment of AM-root colonization, and standard physiological metrics for the determination of plant health 'status. In brief, I showed that the AM symbiosis plays a dynamic role in plant development and stress tolerance first by enhancing the uptake of limiting metal nutrients, and then by reducing the uptake of potentially toxic metal contaminants due to metal biosorption under metal toxicity conditions. Accordingly, I also suggested that such mechanisms contribute in buffering the proximal growth environment, and thereby increasing soil's resiliency. Altogether, I consider the revised models depicting the impact of the AM symbiosis on plant and soil systems to be a relevant tool in environmental remediation practices.

No description available.

Brassinosteroids (BRs) have long been known to be effective in plant growth promotion. However, definitive evidence of BR's role in growth stimulation has remained unclear. Recently, genetic approaches using BR-deficient dwarf(dwf) mutants have begun to unravel the role of BRs in plant growth and development. BR dwarf mutants are characterized by multiple growth alterations: robust stem, reduced fertility, prolonged life cycle, dark-green appearance, round and curled leaves, and when grown in the dark, short hypocotyls and expanded cotyledons. Genetic analysis of the dwf mutants defined eight independent genetic loci defective in BR biosynthesis or perception. Allelism tests with previously reported genes revealed that d̲i̲m̲inuto 1 (dim1) was an allele of dwf1, and dwf2, dwf3, and dwf6 are allelic to b̲r̲assinosteroid i̲nsensitive (bri), c̲onstitutive p̲hotomorphogenesis and d̲warfism (cpd), and d̲e̲-e̲t̲iolated2 (det2), respectively. dwf4, dwf5, dwf7, and dwf8 were found to be novel and are the focus of this research. Anatomical analysis demonstrates that a reduction in cell length causes dwarf phenotype. Dwarfism was rescued by exogenous application of BRs. Feeding studies utilizing BR biosynthetic intermediates were employed to identify defective steps of BR biosynthesis in each of these dwarf mutants. dwf4 mutants were rescued only by 22α hydroxylated BRs, suggesting that the 22α hydroxylation reactions, putative rate-determining steps, are blocked. In fact, DWF4 has been cloned and shown to encode a cytochrome P450 steroid hydroxylase. Feeding studies also showed that dwf8 plants are rescued only by intermediates after 3 dehydrogenation reactions, indicating that the 3-dehydrogenase is defective in dwf8 plants. Gas Chromatography-Selective Ion Monitoring (GC-SIM) analysis of endogenous BRs in dwf5 plants showed that the level of 24-methylene cholesterol is greatly diminished as compared to wild type, suggesting that the biochemical defect occurs before 24-methylene cholesterol. Similar to dwf5, the biosynthetic defect in dwf7 is also shown to be in a step before 24-methylene cholesterol. The pleiotropic phenotypes in these dwf mutants due to biochemical defects in BR biosynthesis suggests that BRs are essential for proper growth and development of plants.

Carotenoids are C₄₀ tetraterpenoids synthesized by nuclear-encoded, multi-enzyme complexes located in the plastids of higher plants. In order to further understand the components and mechanisms involved in carotenoid biosynthesis, our laboratory has identified Arabidopsis thaliana mutants that disrupt this pathway. Here, I report the identification and characterization of three non-allelic albino mutations, pds1, pds2, and pds3 (pds = b phytoene desaturation), that are disrupted in phytoene desaturation, an early step in carotenoid biosynthesis. pds1 and pds2 have been more thoroughly characterized than pds3. Surprisingly, neither pds1 nor pds2 maps to the locus encoding the phytoene desaturase enzyme, indicating the products of at least three loci are required for phytoene desaturation. Electron transport chain components are hypothesized to be involved in phytoene desaturation and the analysis of pds1 and pds2 provide the first genetic evidence that plastoquinone is an essential component in carotenoid biosynthesis. Both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene, affecting distinct steps of the common plastoquinone/tocopherol biosynthetic pathway. The pds1 mutation affects the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) as it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and p-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Additionally, I report the isolation of an Arabidopsis HPPD cDNA, the first from a higher plant, which encodes a 50 kD polypeptide with between 29 and 40% identity to non-plant HPPDs. Alignment of the Arabidopsis HPPD with non-plant HPPDs identifies 38 identical amino acid residues, including six tyrosine and histidine residues thought to form the ferric iron center of the enzyme. When expressed in E. coli, the Arabidopsis HPPD catalyzes the accumulation of two compounds, homogentisic acid and ochronotic pigment, a polymerized oxidation product of homogentisic acid. Additionally, the Arabidopsis HPPD locus and the pds1 locus co-segregate. Finally, and most-significantly, the constitutive expression of the Arabidopsis HPPD cDNA in the pds1 mutant background complements the pds1 mutant. Taken together, these data suggest that pds1 is a mutation in the HPPD gene.