The response of two native Arizona plants, fourwing saltbush (Atriplex canescens) and greasewood (Sarcobatus vermiculatus), to five concentrations of nitrate (tap water only, 50 mg/L, 100 mg/L, 750 mg/L, and 2000 mg/L as nitrate) is investigated. Their growth, transpiration, and nitrate and percent nitrogen tissue concentrations were measured. All of the plants' responses were affected by nitrate concentration. In general, it can be concluded that both fourwing saltbush and greasewood tolerated nitrate concentrations as high as 2000 mg/L. However, greasewood's optimal growth was at Level 4 (750 mg/L nitrate) and its tissue nitrate approximately doubled from an average of 572 ± 255 mg/kg at Level 4 to 1020 ± 511 mg/kg at Level 5 (2000 mg/L nitrate). Fourwing saltbush demonstrated a remarkable ability to tolerate large quantities of nitrate and convert it to organic nitrogen at high concentrations. Fourwing saltbush's largest dry mass, 14.48 ± 2.03 g, was at 2000 mg/L of nitrate.

The auxin indole-3-acetic acid (IAA) is involved in virtually every aspect of plant development. Plants control auxin homeostasis through complex interactions between de novo synthesis, degradation, import, export, and conjugate synthesis and hydrolysis. A thorough knowledge of these pathways and their interactions is key to understanding auxin response and plant growth. I have used genetic and biochemical methods to better understand the function and metabolism of amide-linked conjugates of IAA. Conjugates may function as storage or transport forms of IAA, or may function independently of hydrolysis. Many IAA conjugates have auxin activity in bioassays, and Arabidopsis mutants with reduced sensitivity to exogenous IAA-L-amino acid conjugates have been identified. Some of these are defective in genes encoding conjugate hydrolases, whereas others are likely to be directly or indirectly involved in some other aspect of conjugate metabolism or transport. To better understand conjugate hydrolysis pathways, I have generated and screened an overexpression library to identify cDNAs that lead to conjugate resistance when overexpressed. From this library, I have identified one mutant that cosuppresses petH, resulting in a chlorotic phenotype, and several mutants resistant to IAA-Ala that result from mutations unlinked to T-DNA inserts. In addition, I have cloned the gene defective in iar4, an IAA-alanine-resistant mutant, and found that it encodes a protein similar to a mitochondrial pyruvate dehydrogenase E1alpha subunit. To better understand conjugate hydrolysis, I have characterized the enzyme activities of four members of the amidohydrolase family. I found that bacterially expressed GST fusions of ILR1, ILL2, and IAR3 hydrolyze certain IAA-amino acids with KM values in the muM range, suggesting they are physiologically relevant. In addition, mutant plant extracts show altered rates of conjugate hydrolysis, further supporting the hypothesis that these enzymes function in vivo to cleave IAA-amino acid conjugates. I have also examined the expression profiles of the amidohydrolase family and found their expression is overlapping but distinct. My findings suggest that the activity displayed by auxin conjugates is due to their hydrolysis by the amidohydrolases to yield free IAA, and that these processes are controlled both developmentally and spatially to regulate free IAA levels.

Auxin is an important plant phytohormone that influences virtually all aspects of plant growth and development. Despite the ubiquitous involvement of auxin in plant processes, gaps remain in our knowledge of auxin homeostasis and signal transduction. There are several endogenous forms of auxin including, indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) (Ludwig-Muller and Epstein, 1993). IBA appears to undergo a two-carbon elimination reaction similar to the one used in fatty acid beta-oxidation in peroxisomes to yield IAA (Zolman et al., 2000). To understand the roles of IBA in plants, I have used forward and reverse genetics to isolate IBA-r&barbelow;esponse ( ibr) mutants that display enhanced root elongation on inhibitory concentrations of IBA and yet maintain a wild-type phenotype on IAA. I have categorized these mutants into three distinct classes based on their root and hypocotyl phenotypes under various hormone, sugar, and light conditions. One project involves a mutant, B423, with an exogenous sucrose-dependence for germination, and an inability to produce lateral roots in response to IBA and without hormone. The phenotypes of this mutant suggest a peroxisomal deficiency including defective beta-oxidation of lipid stores and an inability to convert IBA to IAA, necessary for lateral root production. Interestingly, this ibr mutant maps to a region lacking genes known to function in peroxisomal processes. This mutant may define a novel component that has yet to be identified in other organisms. A second project involves two mutants, B705 and B839, which are sucrose independent for germination and produce wild-type numbers of lateral roots in response to IBA. We used positional cloning to determine that one mutant is defective in ACX1 and the other is defective in ACX3, two of the six fatty acyl-CoA oxidase (ACX) genes in Arabidopsis. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third mutant, acx4. Activity assays demonstrated that mutants defective in ACX1, ACX3, and ACX4 have reduced fatty acyl-CoA activity on specific substrates. Moreover, double mutant analysis shows that ACX2 and ACX5 also contribute to IBA response.

The plant cell wall is a complex structure composed in part of cellulose microfibrils interconnected by a network of hemicellulose. The most abundant hemicellulose of dicotyledonous plants is xyloglucan. Xyloglucan endotransglucosylase/hydrolases (XTHs) cleave xyloglucan polymers and religate the newly generated reducing ends to other xyloglucan polymers (Rose et al., 2002). Analysis of the Arabidopsis database reveals an extensive gene family that encodes thirty-three XTH proteins. These proteins are highly similar and share a conserved motif that is predicted to be necessary for their activity, as well as N-linked glycosylation sites and putative signal sequences for translation at ER membranes. Although the biochemical activity of this enzyme is well defined, the physiological consequences of XTH activity in vivo remain undetermined. XTH biochemical activity, however, predicts a role in modification of the cell wall. An attempt was started to elucidate the physiological functions of the 33 XTHs of Arabidopsis. From this work, 47 mutations were identified in 28 XTH genes. Mutations in XTH15 were chosen for detailed study. Theses mutants exhibit a shorter phenotype than wild type when grown at higher temperatures. This difference may be due to the misshapen cell morphology observed in the xth15 mutants through microscopy. XTH gene expression was examine through the use of reporter-gene fusions with the beta-glucuronidase (GUS) gene as well as by compiling evidence of expression from various on-line databases, such as the Genevestigator website. Overall, divergent XTH expression patterns are observed from the earliest stages of seed germination through flowering. XTH::GUS patterns and data from other expression sources show some overlap which may result in novel combinations of XTH activities in distinct cells or organs.

The 57-member family of calmodulin (CaM) and CaM-Like (CML) proteins of Arabidopsis, defined and characterized in this thesis, have a strong potential to serve as sensors and interpreters of calcium signals, which are used in plants to convey information about and elicit specific responses to biotic and abiotic stimuli. Despite this opportunity to affect diverse aspects of plant biology, gene expression patterns or physiological function have been only marginally described for less than 30% of this family. The subset of genes characterized here serve as a primer to a comprehensive analysis of CAM and CML gene expression and function. To examine gene expression, mRNA levels were directly measured for 13 genes using northern analysis or quantitative-PCR (Q-PCR). The CAMs and CMLs tested show distinct, but sometimes overlapping, expression patterns both in induction of expression by stimuli and in localization throughout the plant. Indirect gene expression detection using GUS reporter transgenics allows more precise localization of expression for 15 CAMs and CMLs. Additionally, subcellular fractionation shows that CML12/TCH3 protein may be associated with multiple membranes. Physiological functions of the CAMs and CMLs can be elucidated by analysis of mutants expected to lack CaM or CML proteins. From this work, 50 cam or cml mutants were identified. Mutants chosen for in-depth study, cml11 and cml12/tch3, are among the first described mutations in an Arabidopsis CAM or CML. Root border cells accumulate in cml11, and this difference may contribute to root penetration of media. Although CaM and CML proteins share between 16 and 100 percent identity, it is likely that they have independent functions. CAMs and CMLs show non-redundant spatiotemporal patterns of gene expression, indicating that their proteins may function at different times or locations. Understanding gene expression regulation and physiological function for each CAM and CML will help us understand one aspect of how plants decode calcium signals necessary to complete specific developmental processes or survive changing environmental conditions.

Auxins are an important class of plant hormones implicated in most aspects of plant development, and therefore influence the overall size and shape of a plant. Whereas the signal transduction pathways that sense and respond to auxin remain mysterious, a number of genes do undergo dramatic transcriptional alterations in response to auxin. The Aux/IAA genes were originally isolated based on strong and rapid transcriptional up-regulation following auxin induction. Aux/IAA genes are primary response genes whose products are thought to regulate auxin-responsive transcription. A new member of the Aux/IAA gene family, IAA28, was cloned based on the abnormal auxin responses and unusual auxin-related adult phenotypes of a gain-of-function mutant, including decreased apical dominance and extremely reduced lateral root formation. In addition to auxin, the iaa28-1 mutant is also resistant to inhibition of root elongation by cytokinin and ethylene, but responds normally to other phytohormones. Northern analysis, promoter - reporter gene fusions and Western analysis demonstrate that IAA28 is strongly expressed in roots. IAA28 may be a primary response gene, as IAA28 mRNA levels increase in response to cycloheximide. However, IAA28 transcription decreases upon auxin treatment, a novel response that differs from other characterized members of the Aux/IAA gene family. Experiments with an auxin-inducible reporter construct suggest that IAA28 is a repressor of auxin-induced transcription. Levels of tagged IAA28 protein decrease dramatically after exogenous auxin treatment, thus IAA28 degradation may increase in response to auxin signaling. In addition, microarray analysis suggests that a number of genes have altered transcript levels in the gain-of-function iaa28-1 mutant background. From these findings, IAA28 has been proposed to encode a transcriptional repressor that functions to regulate expression of genes that promote lateral root initiation in response to auxin.

Plants sense their environment and respond through changes in their development and physiology. Mechanical perturbation, darkness, temperature shocks, and exogenous hormones upregulate the expression of the TCH genes in Arabidopsis. The objective of this work was to determine the functions of the TCH3 and TCH4 proteins. TCH3, a calmodulin-related protein with six potential Ca$\sp{2+}$ binding sites, binds Ca$\sp{2+}$, as evidenced by its mobility shift on an SDS-polyacrylamide gel in the presence of Ca$\sp{2+}.$ TCH4, a protein related in sequence to the enzyme xyloglucan endotransglycosylase (XET), has XET activity. The TCH4 XET prefers nonfucosylated oligosaccharide acceptor substrates, has a pH optimum of 6 to 6.5, and has unusually high activity at low temperatures, with an optimum of 12 to 18$\sp\circ$C. To address the role of these proteins in Arabidopsis development and responses to the environment, plants were generated with altered expression of TCH3 or TCH4. These plants were downregulated through antisense RNA, a T-DNA insertion in the gene, or cosuppression. Plants were upregulated by a strong, constitutive promoter driving an exogenous copy of the gene. The plants developed normally and did not show any detectable changes in cellular organization, cell wall integrity, or mechanical strength. Several assays were developed to test the responses of the transgenics to environmental stimuli and hormones. The transgenics responded normally to wind, heat, darkness/low light, gravity, mechanical obstruction, osmotic stress, auxin, and brassinosteroids. However, preliminary evidence suggests that the TCH4 transgenics may respond differentially to cold. Plants with reduced TCH4 levels were more sensitive to low temperatures, and plants overexpressing TCH4 were more hardy at low temperatures. The lack of a pronounced mutant phenotype in the transgenic plants suggests genetic redundancy. Nevertheless, under specific conditions, the TCH3 and TCH4 proteins may have essential functions in Arabidopsis.

Despite their complacent appearance, plants are highly perceptive of stimuli in their environment. Responses to these stimuli enable plants to acclimate to the conditions of their local environment. Fluctuations in cellular calcium (Ca2+) have been implicated as critical signals for transducing external stimuli into intracellular information. How Ca 2+ signals are decoded in plants is largely unknown. The full Arabidopsis genome sequence enabled the identification of a family of 50 genes encoding potential Ca2+ sensors that are related to the quintessential Ca2+-binding protein, calmodulin (CaM). These C&barbelow;aM&barbelow;-l&barbelow;ike or CML proteins have the potential to mediate Ca2+ signaling in plant cells. My thesis work is focused on elucidating the potential physiological significance of two related CML proteins, CML23 and CML24 (also known as TCH2). CML23 and CML24 share over 40% amino acid sequence identity with CaM and have 4 EF-hand Ca2+-binding motifs. CML24 is likely a Ca 2+ sensor because it can undergo Ca2+-dependent changes in hydrophobic interaction chromatography and migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. These characteristics may enable CML24 to interact with, and potentially regulate, targets in a Ca2+-influenced manner. Reverse transcriptase polymerase chain reaction (RT-PCR) and reporter transgenic analysis indicate that CML23 and CML24 expression occurs in all major organs. Quantitative RT-PCR (QRT-PCR) reveals CML24 transcript levels are increased from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid (auxin). In addition, CML24 over- and underexpressing transgenics, cml24 point mutants, and cml23/cml24 double mutants have phenotypes which demonstrate these proteins play roles in flowering time regulation, ion sensitivity, and ABA inhibition of germination and seedling growth. Phenotype analysis also suggests CML23 and CML24 may have a role in de-etiolation (light-induced inhibition of hypocotyl elongation), chlorophyll accumulation, and response to pathogens. Furthermore, a yeast two-hybrid screen suggests CML24 interacts with an autophagy protein. Elucidating CML23 and CML24 expression patterns and physiological functions has provided additional insight into the roles of Ca2+ and Ca2+-binding proteins in plant development and response to the environment.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 68-02, Section: B, page: 0815. Adviser: Hans J. Bohnert. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7031. Adviser: Frederick E. Below. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.