Successful reproduction in plants is a highly-regulated process reliant on the integration of both endogenous and external cues. Different accessions of the model plant Arabidopsis thaliana have been collected, including those with a winter annual or rapid-cycling flowering habit. Natural variation and mutant screens have enabled many flowering time genes to be identified. A key regulator of flowering is FLOWERING LOCUS C (FLC). FLC is a repressor of flowering and is regulated by a number of genes, including those in the autonomous and FRIGIDA-mediated pathways. Of particular interest are FRIGIDA (FRI) and FRIGIDA-LIKE 1 (FRL1) and the autonomous pathway members, FCA and FY. FRI and FRL 1 promote FLC expression making them dominant repressors of flowering. FRI is proposed to initiate chromatin remodelling at the FLC locus leading to increased FLC expression. Once elevated, FLC levels are maintained until plants undergo an extended period of cold, therefore flowering occurs in spring. In contrast, FCA and FY promote flowering by repressing FLC expression. FCA has also been identified as a receptor of the plant hormone abscisic acid (ABA). Upon binding to FCA, ABA is proposed to disrupt/inhibit the FCA:FY interaction which results in delayed flowering. To characterise the FCA:ABA interaction and identify the ABA binding site, a number of truncated FCA proteins were utilised. Initially a FCA:FY GST-pulldown was used to identify the ABA binding site. However, when ABA failed to inhibit the FCA:FY interaction a direct binding assay using [�H]-ABA was employed. Another Arabidopsis ABA receptor, G-protein coupled receptor 2 (GCR2), was used as a positive control in these binding assays. Both FCA and GCR2 failed to bind [�H]-ABA suggesting a broader issue with the binding assay. The identification of FCA and GCR2 as ABA receptors can be attributed to the quality of the protein assayed, the sensitivity of the binding assay and the subsequent data analysis. This study resulted in the retraction of the original paper (Razem et at, 2006) reporting FCA as an ABA receptor. To investigate the molecular mechanism by which FRI and FRL1 act as positive regulators of FLC expresion, a biochemical approach was taken. FRI and FRL1 have no known homology to any other protein or domain and the only method for assessing protein function is through plant complementation experiments. In the absence of sequence homology, or a timely functional assay, a classical approach was taken to produce soluble protein for analysis. Truncation of predicted regions of disorder and expression, solubility and stability screens produced soluble protein of reasonable purity. This allowed characterisation of the biochemical properties of FRI and FRL1. Interaction studies between FRI and FRL1, and the zinc finger protein SUPRESSOR OF FRIGIDA 4 (SUF4), were also carried out. Polyclonal antibodies against FRI and FRL1, made during this study, were useful for protein detection in these experiments. The interaction studies, together with plant complementation experiments, suggest that the C-terminus of FRI is essential for protein function, while the N-terminus improves FRI activity. These findings provide a better understanding of how the components of the proposed "FRI-complex" may interact to promote FLC expression.

Bibliography: leaves 89-104. In this study an EMS-mutated Arabidopsis mutant pho2, which accumulates Pi in leaves, was used to study Pi uptake and transport by comparing it to wild-type seedlings. The study aimed to define the physiological lesions in pho2 mutant and to obtain evidence regarding the function of the PHO2 gene in P nutrition in higher plants. Accumulation of Pi in leaves of pho2 was found to reside in the symplast and was not related to Zn-deficiency. The physiology of the pho2 mutant is consistent with either a block in Pi transport in phloem from shoots to roots or an inability of shoot cells to regulate internal Pi concentration. Southern block analysis revealed that the two transporter genes, APT1 and APT2 were not responsible for the pho2 mutant. Data from the mapping of the PHO2 gene along with information from the Arabidopsis genome sequencing will form the basis for cloning the PHO2 gene in the future.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 103-124).

The GABI Primary Database, GabiPD (http:// www.gabipd.org/), was established in the frame of the German initiative for Genome Analysis of the Plant Biological System (GABI). The goal of GabiPD is to collect, integrate, analyze and visualize primary information from GABI projects. GabiPD constitutes a repository and analysis platform for a wide array of heterogeneous data from high-throughput experiments in several plant species. Data from different ‘omics’ fronts are incorporated (i.e. genomics, transcriptomics, proteomics and metabolomics), originating from 14 different model or crop species. We have developed the concept of GreenCards for textbased retrieval of all data types in GabiPD (e.g. clones, genes, mutant lines). All data types point to a central Gene GreenCard, where gene information is integrated from genome projects or NCBI UniGene sets. The centralized Gene GreenCard allows visualizing ESTs aligned to annotated transcripts as well as displaying identified protein domains and gene structure. Moreover, GabiPD makes available interactive genetic maps from potato and barley, and protein 2DE gels from Arabidopsis thaliana and Brassica napus. Gene expression and metabolic-profiling data can be visualized through MapManWeb. By the integration of complex data in a framework of existing knowledge, GabiPD provides new insights and allows for new interpretations of the data.

<p>In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised.</p>

Chloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin and to have been transferred from a symbiont's genome to host genome via lateral transfer. Subsequent duplication of the initial FtsZ gene gave rise to the FtsZ1 and FtsZ2 genes and protein families in eukaryotes. These proteins co-localize mid-chloroplast to form the Z-ring. Z-ring assembly initiates chloroplast division, and it serves as a scaffold for other chloroplast division proteins. Little is known, however, about the FtsZ protein subunit turnover within the Z-ring, the effects of accessory proteins on Z-ring turnover assemblies, as well as the in vivo ultrastructure of the Z-ring in plants. To investigate the Arabidopsis thaliana FtsZ subunit turnover rate within the Z-ring, a section of the Z-ring in the chloroplasts of living plants expressing fluorescently tagged FtsZ1 or FtsZ2 proteins was photobleached and the recovery rate was monitored. The results show that the fluorescence recovery half times for the FtsZ1 and FtsZ2 proteins are 117s and 325s, respectively. This is significant as these data mirror their differences in GTP hydrolysis rates. To elucidate in vivo structure and ultrastructure of the Z-ring, a protocol was established that maintained fluorescence during high pressure freezing, freeze substitution and low temperature embedding. Afterwards, a correlative microscopy approach was employed to visualize and identify fluorescently labeled puncta, circular structures, at the light microscopy level. These puncta were further resolved as mini-rings using optical microscopy eXperimental (OMX) superresolution microscopy. Electron microscopy (EM) analysis imaged mini-rings and filament assemblies comprised of dense subunits. Electron tomography (ET) showed mini-rings composed of protofilaments.

How patterns arise from an apparently uniform group of cells is one of the classical problems in developmental biology. The mechanism is complicated by the fact that patterning occurs on a growing medium. Therefore, changes in an organism’s size and shape affect the patterning processes. In turn, patterning itself may affect growth. This interaction between growth and patterning leads to the generation of complex shapes and structures from simpler ones. Studying such interactions requires the possibility to monitor both processes in vivo. To this end, we developed a new technique to monitor and quantify vein patterning in a growing leaf over time using the leaves of Arabidopsis thaliana as a model system. We used a transgenic line with fluorescent markers associated with the venation. Individual leaves are followed in many samples in vivo through time-lapse imaging. Custom-made software allowed us to extract the leaf surface and vein pattern from images of each leaf at each time point. Then average spatial maps from multiple samples that were generated revealed spatio-temporal gradients. Our quantitative description of wild type vein patterns during leaf development revealed that there is no constant size at which a part of tissue enclosed by vasculature will become irrigated by a new vein. Instead, it seemed that vein formation depends on the growth rate of the tissue. This is the first time that vein patterning in growing leaves was quantified. The techniques developed will later be used to explore the interaction between growth and patterning through a variety of approaches, including mutant analysis, pharmacological treatments and variation of environmental conditions.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

How patterns arise from an apparently uniform group of cells is one of the classical problems in developmental biology. The mechanism is complicated by the fact that patterning occurs on a growing medium. Therefore, changes in an organism’s size and shape affect the patterning processes. In turn, patterning itself may affect growth. This interaction between growth and patterning leads to the generation of complex shapes and structures from simpler ones. Studying such interactions requires the possibility to monitor both processes in vivo. To this end, we developed a new technique to monitor and quantify vein patterning in a growing leaf over time using the leaves of Arabidopsis thaliana as a model system. We used a transgenic line with fluorescent markers associated with the venation. Individual leaves are followed in many samples in vivo through time-lapse imaging. Custom-made software allowed us to extract the leaf surface and vein pattern from images of each leaf at each time point. Then average spatial maps from multiple samples that were generated revealed spatio-temporal gradients. Our quantitative description of wild type vein patterns during leaf development revealed that there is no constant size at which a part of tissue enclosed by vasculature will become irrigated by a new vein. Instead, it seemed that vein formation depends on the growth rate of the tissue. This is the first time that vein patterning in growing leaves was quantified. The techniques developed will later be used to explore the interaction between growth and patterning through a variety of approaches, including mutant analysis, pharmacological treatments and variation of environmental conditions.