The proper distribution of proteins is important for the development and function of both individual cells and whole organisms. Relatively little is known about the mechanisms of protein localization in plant cells. Protein body formation in maize endosperm provides a useful system in which to study these mechanisms. Maize endosperm is a specialized tissue that accumulates starch and storage proteins and ultimately provides nutrients for the germinating seedling. The storage proteins, called zeins, are cotranslationally inserted into the rough endoplasmic reticulum (RER) where they aggregate into spherical protein bodies. Previous studies have suggested that the cytoskeleton may play a role in protein body formation, since actin, the protein synthesis factor EF-1α (which associates with the cytoskeleton in other systems), and polysomes, including zein polysomes, were found associated with protein bodies following endosperm homogenization. To determine whether the cytoskeleton, EF-1α, and protein bodies are associated in situ, these components were visualized in intact endosperm cells. By using indirect immunofluorescence and confocal microscopy, changes were documented in the distributions of EF-1α, actin, and microtubules during development. The protein bodies are found enmeshed in EF-1α and actin and are juxtaposed with a multidirectional array of microtubules. In addition, actin and EF-1α appear to exist in a complex. Finally, actin bundling assays demonstrated that maize EF-1α is capable of bundling actin in vitro. Therefore, EF-1α may organize actin around protein bodies. One possible role of the cytoskeletal network around protein bodies is the transport and or anchoring of zein mRNAs to sites of protein body formation. To test this hypothesis, in situ hybridization experiments were conducted in the presence and absence of cytoskeleton disrupting agents. The results suggest that while the zein mRNAs may associate with the RER at random sites, a cytoskeleton-dependent transport mechanism may be utilized to traffick them to the RER surface. To more convincingly demonstrate a role of the cytoskeleton in zein mRNA localization, microinjection and visualization of zein mRNAs in the presence and absence of intact cytoskeletal elements is required. Methods for microinjecting maize endosperm cells with such mRNAs were devised and preliminary results are described.

Polyhydroxylated steroids with insect molting hormone activity were discovered in plants over thirty years ago. The major endogenous molting hormone of insects is believed to be 20-hydroxyecdysone (20E) and interestingly, it is also the most commonly encountered phytoecdysteroid (PE) in plants. Ecdysteroids control developmental programs in both immature and adult insects however, the role of PEs in plants has not been demonstrated. PEs are hypothesized to function as either plant hormones or plant defenses against phytophagous insects. Many toxic secondary metabolites are concentrated in apical meristems where herbivory would result in the greatest reduction in plant fitness. Similarly, the highest concentrations of 20E in spinach were associated with the stems and vasculature while old leaves and roots displayed low levels. In plants, concentrations of toxic or deterrent metabolites are often rapidly induced following attack. In spinach roots, both mechanical damage and insect herbivory resulted in rapid increases in 20E concentrations. The plant wound signal, jasmonic acid was strongly implicated in signaling this response. Known plant hormones and chemical defenses are regulated differently. Pulse chase studies with [2-¹⁴C] mevalonic acid demonstrated that de novo root 20E biosynthesis occurred during the induction and, once synthesized, 20E was stable for over one month. This result is does not support the plant hormone hypothesis, as plant hormones typically undergo rapid conjugation or catabolism. The induction of root 20E concentrations occurred without similar changes in related membrane phytosterols. Simply, pathway specificity was demonstrated as increased 20E accumulation was not part of an overall increase in steroids. To empirically examine the hypothesis that PEs function as plant defenses against insects, a series of experiments were designed with the fungus gnat Bradysia impatiens. Results indicated that root herbivory by larvae induced 20E levels in roots, larval preference for diets containing induced 20E levels was reduced, larval survivorship on 20E containing diets was lower, and plants with induced root 20E levels were better protected from attack. Together, these results support the plant defense hypothesis at both the physiological and ecological level.

Microarray analysis and mutational approaches were used to investigate regulation of freezing tolerance in plants. Using Arabidopsis, cold-responsive gene expression profiling was performed with Affymetrix GeneChip. Arabidopsis seedlings were cold-treated at 0°C for 0, 3, 6, and 24 hrs. A total of 681 cold-responsive genes were identified. Comparison of the expression profile of cold-responsive genes in the wild type to that of ice1, a mutant defective in cold stress signaling, showed altered transcript levels for many cold-responsive genes in the ice1 mutant even under non-stress conditions, which may explain its altered sensitivity to cold stress. To dissect the regulatory mechanism of cold-responsive gene expression, Arabidopsis plants expressing the luciferase gene driven by stress-responsive promoters were mutated and screened for altered luminescence. fro1 (frostbite 1),fro2 (frostbite 2), and stab1 (stabilized 1) were characterized and the genes responsible for the mutant phenotypes were identified. fro1 showed a lower expression of the stress-responsive RD29A promoter-driven luciferase transgene and endogenous RD29A gene under cold than the wild type. FRO1 encodes a component of complex I in the mitochondrial electron transport chain. These results suggested that cold-inducible gene expression is modulated by the functional status of mitochondria. fro2 was identified from Arabidopsis expressing the CBF3 promoter driven-luciferase transgene, because of its lower luminescence induction under cold. fro2 also showed lower expression of endogenous CBF genes under cold. FRO2 encodes a serine decarboxylase that converts serine to ethanolamine. This result indicated the importance of serine metabolism in CBF gene regulation. stab1 showed higher expression of RD29A promoter-controlled luciferase after stress, but the level of endogenous RD29A transcript was not different from that of the wild type after stress. Nuclear run-on assays suggested that this discrepancy was due to the enhanced stability of the luciferase mRNA. STAB1 encodes a splicing factor similar to the human U5 snRNP-associated 102-kDa protein. stab1 displayed hypersensitivity to cold and altered sensitivity to various stress. These results suggested that STAB1 is required for not only splicing but also the turnover of unstable transcripts and that it has an important role in plant tolerance to abiotic stresses, particularly cold.

The hexagons on a pineapple contrast with the ribs observed, for example, on pumpkins or saguaro cacti. This dissertation demonstrates how these various configurations, and also the related patterns of phyllotaxis (the arrangement of leaves into whorls or spirals) can be understood as the energy-minimizing buckling pattern of a compressed shell (the plant's tunica) on an elastic foundation. The key new idea is that the elastic energy is minimized by special triads or sequences of triads of periodic deformations whose local wavevectors add to zero. Although triad configurations arise from a variety of microscopic mechanisms in natural and laboratory systems, we show that the particular choices of wavevectors that are observed on plants arise in a nontrivial way from properties specific to a mechanical model. Furthermore, the theory predicts correlations between types of phyllotaxis and shapes of plant surface configurations and suggests experiments that can further test the mechanical theory of plant pattern formation. The dissertation concludes with a derivation of Cross-Newell equations governing pattern formation far from onset in nonisotropic systems and in systems with hexagonal planforms.

Little is known about the crown system and its association with plant growth and development in spring cereals. This study investigated temperature, seeding depth and genotypic effects on crown development of barley; relationships between crown and seminal root systems; and classification and description of crown systems under deep seeding. Two greenhouse experiments were performed using PVC tubes. Low temperature and deep seeding reduced percentage and rate of emergence but increased crown number, crown depth, and crown weight in most genotypes. Crown number, crown depth and crown weight showed increased associations with seminal root, whole root, and shoot weight at low temperature and deep seeding in most genotypes. Deep plantings showed that crown systems of barley can be classified as unicrown, bicrown and multicrown types with some variants. One line produced plants with no subcrown internode under 12.5 cm planting. Our results suggested that the crown is a potential source of crown roots and tillers.

Studies were performed to determine the concentration of soluble carbohydrates and starch in chrysanthemum plants at various stages of vegetative and floral development. Leaves of pot chrysanthemum (Chrysanthemum morifolium 'Charm' or 'Favor') grown under full irradiance had more soluble carbohydrates and starch than those grown with 65% irradiance reduction. Both showed clear diurnal variation in carbohydrate concentrations. Inflorescences exhibited no diurnal fluctuations in total soluble carbohydrate (TSC). Sucrose was the only translocated carbohydrate in chrysanthemums in quantities detectable by HPLC. In a postproduction environment, leaf and stem TSC remained relatively unchanged while inflorescence TSC decreased significantly. Reducing sugars (glucose + fructose) accounted for up to 84% of the inflorescence TSC. Relative levels of starch and fructans over time suggests an alternate use of fructans and starch as pools of available reserve carbohydrate during floral development. Fructans were shown to decrease in polymerization in both petals and inflorescences as petals expanded.

Recent investigations indicate that 2,4,6-trinitrotoluene (TNT) is rapidly transformed in an aquatic environment by the action of photosynthetic organisms. Several plant species from a variety of culture conditions were examined that displayed the capacity to transform TNT. The kinetics of transformation and the fate of transformation products were monitored by MECE and HPLC analysis in both the aquatic media and within the plant tissues. (U)$\sp{14}$C-TNT was introduced into the plant systems in order to monitor the fate of the aromatic ring. In order to assess the action of plant biochemistry in isolation from ordinary periphyton, axenic plants were examined for their metabolic capacity. Experiments showed that the majority of TNT is rapidly transformed into an unidentified soluble product in the medium and plant tissues with small quantities of reduction products appeared transiently. Over time, an increasing amount of $\sp{14}$C was irreversibly associated with the plant tissue.

Auxins are a family of hormones that influence numerous aspects of plant growth and development, including apical dominance, vascular development, tropic responses, inhibition of root elongation, and initiation of lateral roots. Indole-3-butyric acid (IBA) is a naturally-occurring auxin that efficiently induces rooting and is widely used in commercial and agricultural settings. Interestingly, one mechanism of IBA action is via its conversion to the more abundant auxin indole-3-acetic acid (IAA) in a pathway similar to fatty acid beta-oxidation. This reaction should occur in peroxisomes, which are small organelles that contain enzymes required for fatty acid beta-oxidation, the glyoxylate cycle, and branched-chain amino acid catabolism. More than 20 peroxin (PEX) proteins are required for peroxisomal biogenesis and maintenance. To better understand the in vivo role of this naturally-occurring auxin, we have identified a collection of Arabidopsis thaliana mutants that are resistant to the inhibitory effects of IBA on root elongation, but that remain sensitive to IAA. These mutants have defects in various IBA-mediated responses, which allowed us to group them into five phenotypic classes. Defects in seedling development in the absence of exogenous sucrose suggest that some of these mutants have defects in the beta-oxidation of seed storage lipids, implying that the conversion of IBA to IAA also is disrupted. Other mutants appear to have normal peroxisomal function; these may be defective in IBA transport, signaling, or responses. We have used positional information to clone the genes defective in eight distinct loci. Three mutants, pex5, pex6, and pxa1, have defects in proteins expected to act in peroxisomal biogenesis or import. In addition, we have isolated mutants with defects in the medium-chain acyl-CoA oxidase ACX3 and in two peroxisomal acyl-CoA dehydrogenases with unknown functions. Lastly, we have identified a mutant defective in CHY1, which encodes an acyl-CoA hydrolase that may act in peroxisomal valine catabolism. These results indicate that in Arabidopsis, IBA acts, at least in part, via its conversion to IAA. Moreover, IBA resistance is a powerful tool to identify genes acting in peroxisomal beta-oxidation, providing an unbiased approach for studying peroxisomal function in plants.

Humankind depends on plants to harvest solar energy and convert it into accessible chemical energy. With booming human population growth and diminishing availability of arable land, understanding plant development is necessary for more efficient agricultural production. Auxin is a plant hormone utilized in many aspects of plant growth and environmental responses. This work examines genes that regulate or are regulated by auxin, biogenesis and function of an organelle that is an auxin source, and molecules that behave as auxins to influence plant development. Within the plant cell, peroxisomes are organelles that house many processes including fatty acid metabolism to produce energy and also proto-auxin metabolism to produce the active hormone. Peroxisomal proteins are translated in the cytoplasm and imported into peroxisomes by a host of machinery. Peroxisomal targeting signal sequences are recognized by one of two receptors; these receptors interact with each other physically and functionally in some organisms. Here, I identify the receptor machinery present in diverse organisms to predict and compare methods of peroxisomal matrix protein import. I also characterize mutants of the model plant Arabidopsis thaliana defective in import of one class of peroxisomal matrix proteins. In addition, I examine various molecules that influence plant development in an auxin-like fashion. I identify genes with mRNA accumulation regulated by a proto-auxin in a background that inefficiently converts this compound into auxin. I describe the characterization of responses to a second auxin-related molecule that impacts plant development through auxin signaling. I also describe the isolation, characterization, and cloning of a mutant with reduced sensitivity to a specific subset of auxin-like molecules. Data obtained in this work reveal a host of factors that affect auxin regulation and thereby influence plant life. The results of these experiments in plant biology highlight the diversity, complexity, and essentiality of auxin responses.

The objectives of this research were to study metabolic pathways in C. roseus hairy root cultures, and to enhance secondary metabolite productivity using fungal elicitation as the technique. The effects of age of inoculum were studied by adapting the cultures to three subculture cycle routines. The 2 week subculture cycle yielded the fastest, while the 4 week cycle yielded the lowest, specific growth rates. Specific yields of tabersonine decreased from day 21 to 35 while the total yields of horhammericine increased in all three subculture cycles. Lochnericine yields were highest in the 2 week cycle while serpentine yields were lowest. The effects of dosages and exposure times of specific elicitors on several compounds in the indole alkaloid pathway were studied. A 150% increase in tabersonine specific yield was observed upon addition of 72 units of pectinase. Levels of serpentine, tabersonine and lochnericine decreased transiently after addition of pectinase in time course studies. Jasmonic acid was found to be a unique elicitor leading to an enhancement in flux to several branches in the alkaloid pathway. Time course studies with jasmonic acid showed a transient increase in lochnericine and tabersonine levels and a continuous increase in levels of ajmalicine, serpentine and horhammericine. NMR spectroscopy was utilized as the tool to study primary metabolism of hairy roots non-invasively. $\sp{31}$P NMR spectroscopy studies indicated that vacuolar and cytoplasmic pH were maintained at 7.4 $\pm$ 0.06 and 5.25 $\pm$ 0.08 respectively. $\sp{13}$C NMR spectroscopy studies indicated activities of pentose phosphate pathways, non-photosynthetic CO$\sb2$ fixation and glucan synthesis pathways. Recycling of triose phosphate was evident from scrambling of label in glucans. In vivo $\sp{31}$P and $\sp{13}$C NMR spectroscopy was subsequently utilized to study the effects of elicitors on primary metabolism. A transient short-term decline in the cytoplasmic pH was observed upon addition of pectinase while a prolonged decrease in vacuolar pH was observed upon addition of jasmonic acid. Enhanced accumulation of glucans was detected upon addition of pectinase. A reduction in the levels of pyruvate and glutamine was observed, upon addition of jasmonic acid, indicating a decrease in flux to glycolysis or an increase in the drain on these pools.