Abscisic acid (ABA) is a plant hormone that regulates various developmental processes and environmental stress responses. ABA modulates growth of both primary roots and lateral roots, helping to shape root architecture. The lateral root organ defective (latd) mutants, disrupted in the MtLATD/NIP gene, encoding a nitrate transporter, have severe root growth defects that can be rescued by applying ABA. However, the way in which ABA stimulates latd root growth is unclear, and the downstream components of MtLATD/NIP and ABA signaling are completely unknown. To answer these questions, this dissertation focuses on two major potential downstream regulators: Reactive Oxygen Species (ROS) and transcription factors (TFs). ROS are important signaling molecules required in ABA-induced stomatal closure under drought or osmotic stresses, but their role in ABA regulation of root development is unclear. I found that latd mutant roots have increased ROS levels, and the expression level of several MtRboh genes, which encode major ROS-producing enzymes, the NADPH oxidases, is also increased. ABA decreases the amount of ROS in latd roots and also reduces expression of MtRbohC, in particular. In addition, I observed that latd mutant roots have cell elongation defects, which can also be rescued by exogenous ABA. I demonstrated that pharmaceutically decreasing ROS levels using an NADPH oxidase inhibitor, or reducing the expression of MtRbohC using RNA interference can increase cell elongation and stimulate lateral root elongation in latd roots. These findings have revealed a mechanism by which ABA restores root growth in latd mutant roots via regulating ROS levels, and identified MtRbohC as an important downstream target of ABA signaling mediated by MtLATD/NIP. TFs act as regulatory nodes controlling the transcription of gene clusters and playing a crucial role in plant growth and development. Using a high-throughput TF profiling approach, I have identified 20 TFs that exhibit altered expression levels in latd mutant roots as compared to wild type, 60% of which can be restored to normal levels by ABA. My analysis also revealed that ABA regulates the expression of a different set of TFs in latd roots, suggesting that MtLATD/NIP is crucial for ABA regulation of TF expression. Moreover, ABA changes the TFs regulated by MtLATD/NIP almost completely, indicating a tight control of ABA on TFs regulated by MtLATD/NIP. Surprisingly, I found that the expression of NODULATION SIGNALING PATHWAY 2 (MtNSP2), a GRAS family TF required for nodulation, is regulated by MtLATD/NIP, ABA and nitrate in non-symbiotic roots. In symbiotic roots, MtLATD/NIP is required for the transcriptional signaling pathway downstream of MtNSP2 in the epidermis as well as induction of MtNSP2 expression by cytokinin and subsequent activation of its downstream targets in the cortex. These findings indicate that MtLATD/NIP functions in nodulation signal transduction upstream of MtNSP2, and mediates crosstalk with cytokinin. Together, these two approaches have begun to characterize a signaling pathway downstream of ABA and MtLATD/NIP that involves ROS, MtNSP2, and a core group of TFs in the regulation of root development and nodulation in M. truncatula.

Phylogenetic analysis is a powerful tool for elucidate evolutionary relationships of organisms and genes and for testing taxonomic and evolutionary hypotheses. I conducted phylogenetic analyses of DNA sequences from five gene regions to evaluate the classification scheme of Philadelphus and used phylogenetic analysis to provide a framework for examining molecular evolution of the LFY gene in plants. Results from my study suggested that Philadelphus is a paraphyletic group with the single species genus Carpenteria nested within. Three evolutionary distinct clades were identified in this large Carpenteria-Philadelphus complex, the subgenus Deutzoides clade, the genus Carpenteria clade, and the remaining Core-Philadelphus clade, each merits the recognition of a genus. Our results mostly agreed with the most recent treatment of genus Philadelphus on the placement of Deutzoides, with the exception of P. hirsutus. However, our result does not support the classification scheme proposed for the rest Philadelphus species. Biogeographic analysis using the Statistical Bayes-DIVA method (S-DIVA) and divergence time dating with the BEAST method resolved the origin of Philadelphus s. l. in southwestern North America in the Oligocene. Several intercontinental migrations from North America to Asia and to Europe occurred at the different times of the later Tertiary to reach a worldwide distribution of the genus. For the molecular evolution study of LFY gene, our results revealed that the evolution of LFY was under strong functional constraint, with the C domain under the strongest selection force and the intervening domain being the most relaxed. Our study also showed that the detection of positive selection using the Branch Site Model was robust to taxon sampling density, but sensitive to sequence length and alignment ambiguity. Our analyses under various conditions consistently detected positive selection in Fabaceae, where FLO/LFY evolved a role of the KNOX1 gene function in regulating compound leaf development. Under the best alignment, we detected adaptive selection at several sites in Asterales, Brassicaceae, and Fabaceae where gene duplication and/or novel function of LFY have been reported.

The tropical savanna-forest boundary is commonly characterized by an abrupt transition in vegetation structure and in tree species composition. It has been hypothesized that differences in architecture between savanna and forest trees have an important role in determining the contrasting structural differences between savanna and forest ecosystems. Because of the importance the vegetation structure in determining the ecosystem properties of these systems, I performed a comparative study of tree architecture to examine differences in plant structure of savanna and forest species. To eliminate the potential bias from phylogenetic relatedness, I used congeneric species pairs containing trees of both habitat types that occur sympatrically in savannas of the Brazilian cerrado habitat at IBGE Ecological Reserve (RECOR). I found that relative to savanna species, forest species have larger crown volumes with more apical meristems and greater height for a given stem diameter. Other traits that influence patterns of light interception also differed, with savanna species exhibiting more convoluted leaf blades and shorter petioles. There was evidence that allometry and other traits are convergent in savanna and forest tree species across lineages, providing strong support for adaptive functions of these traits. Furthermore, the larger canopies of forest species imply that they play a role in reduced light in the understory and the exclusion of grasses, which potentially facilitates further expansion of forest tree species in the absence of fire.

Anthocyanins are one of the richest pigments, which belong to flavonoid compounds in plant kingdom. They have many biological and ecological functions. Over the past many years, numerous efforts have been made to determine the biosynthetic pathway of anthocyanins and also to identify several regulatory proteins mainly in flowers and fruits of model plants and crop plants. However, many questions concerning the metabolism of anthocyanins in foliage remains unsolved. One example is âHow can developmental processes impact on accumulation patterns of anthocyanins in leavesâ. In this study, we choose several cultivars from one of the most popular ornamental plants Acer palmatum Thunb. to understand the mechanism of developmental changes of pigmentation in leaf. Several other maple species were also analyzed. We propose that the metabolism of anthocyanins play an essential role in such changes. We use an integrated approach of phytochemistry and metabolic profiling to determine the biosynthesis and metabolism of anthocyanins and their impacts on foliage color. Proanthocyanidin analysis was carried out as well to determine their relationship to both anthocyanin production and foliar coloration. We have found that even for green leaves with no/trace amount of detectable anthocyanins, the biosynthetic pathway of anthocyanidin/proanthocyanidin is still activated. Our results indicate that metabolic channeling directing the anthocyanin pathway to the proanthocyanidin biosynthesis plays a very important role in pigmentation pattern change along developmental processes.

<p>Root system architecture (RSA) is the spatial distribution of roots of individual plants. As part of a collaborative effort I adapted a gellan gum based system for imaging and phenotyping of root systems in maize. This system was first used to perform a survey of 26 distinct maize varieties of the Nested Association Mapping (NAM) population. The analysis of these data showed a large amount of variation between different RSA, in particular demonstrating tradeoffs between architectures favoring sparse, but far reaching, root networks versus those favoring small but dense root networks. To study this further I imaged and phenotyped the B73 (compact) x Ki3 (exploratory) mapping population. These data were used to map 102 quantitative trait loci (QTL). A large portion of these QTL had large, ranging from 5.48% to 23.8%. Majority of these QTLs were grouped into 9 clusters across the genome, with each cluster favoring either the compact of exploratory RSA. In summary, our study demonstrates the power of the gellan based system to locate loci controlling root system architecture of maize, by combining rapid and highly detailed imaging techniques with semi-automated computation phenotyping.</p> / Dissertation

Nuclear-encoded chloroplast proteins are synthesized as precursors in the cytosol with N-terminal cleavable transit peptides. The post translational import of proteins into chloroplasts occurs first through the outer membrane via the Toc complex (Translocon of Outer membrane of Chloroplast). The high fidelity of the protein import process is maintained by the specific recognition of the transit peptide of nucleus-encoded proteins by the coordinate activities of two homologous GTPase Toc receptors, Toc34 and Toc159. Structural and biochemical studies suggest that dimerization of the Toc receptors functions as a component of the mechanism to control access of preproteins to the membrane translocation channel of the translocon chloroplast envelope. I show that specific mutations that disrupt receptor dimerization in vitro reduce the rate of protein import in transgenic Arabidopsis compared to the wild type receptor. The mutations do not affect the GTPase activities of the receptors. Interestingly, these mutations do not disrupt initial preprotein binding at the receptors, but they reduce the efficiency of the transition from preprotein binding to membrane translocation. These data indicate that dimerization of receptors has a direct role in protein import, and support a hypothesis in which conformational changes that initiate membrane translocation of chloroplast preproteins is part of the molecular mechanism of GTP-regulated protein import.

Natural climate solutions, such as reforestation, are increasingly called for to lower atmospheric CO2 concentrations and prevent further warming of the climate. Predictive modeling of forest stand dynamics provides a quantitative framework that can be used to select the tree species with the highest carbon (C) uptake potential for restoration efforts based upon site-specific and species-specific data. To simulate stand dynamics and compare C uptake and storage potential of three pine species across Florida, I developed individual-based models that combined environmentally-sensitive tree growth models with background mortality taken from the literature. Growth model parameters were estimated using space for time (SFT) substitution and mortality model parameters were estimated from published literature possibly introducing biases into model development. Therefore, to explore these possible biases, parameters of both growth and mortality models were calibrated through a Bayesian inversion technique using forest growth and mortality data. Simulated results of total above-ground biomass (AGB), for both calibrated and SFT models, were compared with Forest Inventory and Analysis (FIA) re-measurement data. Model results demonstrate that SFT substitution adequately predicted growth rate of P. taeda, P. palustris, and P. elliottii, while Bayesian inversion helped to calibrate parameters in mortality functions reported in published literature. The results highlight the possible benefit of using SFT substitution in tree growth models, helping to save time and resources, as this modeling framework can be easily replicated for forests in other states using open-sourced data from FIA and globally gridded raster data of climate and edaphic properties.

To understand the molecular mechanisms that regulate expansion in plants, I analyzed a mutant, rsw4, previously isolated on the basis of temperature-dependent root swelling. Map based cloning had identified RSW4 as At4g22970, which contains a domain homologous to separase, an enzyme demonstrated to be required for sister chromatid disjunction during mitosis in animals and fungi. I confirmed a role for separase in segregation of sister chromatids in plants. However, with defects in chromosome disjunction, I detected no DNA damage. In addition, the non-disjoined chromosomes did not trigger detectable cell cycle arrest, based on the expression of cell cycle marker genes examined by real time qRT-PCR. Interestingly, the degradation of cyclin B1;1 was prevented in rsw4, based on the extensive accumulation of a reporter construct based on GUS. The transverse alignment of cortical microtubules, which are widely believed to define the directionality of expansion, were disrupted in rsw4 . The disorganization of cortical microtubules was concomitant with root swelling. In addition, polarity of cortex cells appeared to be affected in rsw4. The polar targeting of the auxin efflux protein, PIN2, reversed polarity in cortex cells of rsw4. One micro-array run showed a variety of genes with changed expression level in rsw4. By real rime PCR, I confirmed some genes to have altered expression, including enzymes involved in cell wall metabolism, proteins involved in ethylene signaling, and proteins related to calcium signaling. The finding of changes in the expression of these genes provides potential connections between defective mitosis and aberrant expansion. This study not only confirmed the conserved roles of plant separase in chromosome disjunction, but also advanced our understanding of regulation of plant cell cycle and expansion. My results and following research should provide further insight into the relationship between chromosome disjunction, cell cycle regulation, and expansion.

Rocky Canyon Quarry is a granite mine located in the Santa Lucia Mountains about 5 miles southeast of the city of Atascadero. The Surface Mining and Reclamation Act (SMARA), which passed in 1975, dictates that California mines must rehabilitate lands disturbed by mining operations. Dr. V. L. Holland and his students in the Biological Sciences Department at California Polytechnic State University, San Luis Obispo (Cal Poly) have been working on restoration and revegetation of the Rocky Canyon Quarry since the early 1990’s. Adenostoma fasciculatum (chamise) dominates the chaparral communities (chamisal chaparral) found in Rocky Canyon. Chamise is the most common shrub in much of California’s chaparral and is adapted to recovery after fires by both seed germination and lignotuber sprouting. In laboratory and greenhouse experiments, it has been shown that chamise seeds have an increased germination rate when subjected to fire related stimulates such as heat, smoke, and charate. The goal of my research, which was conducted over a two year period (2002-2004), is to find successful, inexpensive techniques to restore chamisal chaparral on the mined granite slopes of Rocky Canyon Quarry. My study focused on examining and comparing the different techniques that have been shown by other researchers in laboratory and greenhouse situations to increase the germination rate of chamise. I compared the effects of heat, sulfuric acid, powdered charred wood (charate), and smoke on the germination of chamise seeds under the natural field conditions found in the Rocky Canyon Granite Quarry. Seeds were subjected to between one and four of these treatments, to examine possible interactive effects amongst the treatments, and then placed in plots containing the topsoil used to restore the mined hillsides. Plots remained under natural field conditions, and seedlings were counted following emergence. Results of my studies indicate that the smoke related treatments of liquid smoke and chamise smoke have the greatest effect in increasing the seed germination rate of chamise and that there is little to no benefit in subjecting chamise seeds to more than one treatment.

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